Detection of compounds that affect therapeutic activity

Abstract

The present invention relates to methods of detecting compounds that affect the activity of a therapeutic substance or composition administered to a subject, and to reagents for use in such methods.

Description

BACKGROUND OF THE INVENTION

Subjects being treated with therapeutic substances or compositions may experience changes in the activity or the effectiveness of the therapeutic substance because of the presence of certain compounds in the subject. For example, the administration of a therapeutic substance may result in the formation of antibodies against that therapeutic substance by the subject to whom the therapeutic substance was administered. In particular, if such anti-therapeutic antibodies are neutralizing antibodies that prevent the beneficial activity of the therapeutic substance, this phenomenon can have an adverse effect on the treatment of the subject.

Cytokines or growth factors exert their biologic effects by binding to their receptors and activating various intracellular signal transduction processes (Schlessinger and Ullrich (1992) Neuron 9: 383-391; Kishimoto et al. (1994) Cell 76: 253-262; Ihle (1995) Nature 377: 591-594; Wells (1996), Proc. Natl. Acad. Sci. USA 93: 1-6; Dhanasekaran (1998), Oncogene 17: 1329-1330). The synergistic action of the activated intracellular signaling pathways causes alterations in gene expression and further leads to changes in cell survival, proliferation or apoptosis (Kishimoto et al. (1994); Ihle (1995); Appleby et al., (1996) Cell 86: 845-848; Dhanasekaran 1998). These changes reflecting the biologic effects of the growth factors or cytokines have been widely used as biomarkers in existing cell-based bioassays for determining the quantities of biologically active cytokines or growth factors (Mire-Sluis (2001) Pharm. Research 18: 1239-46; Eghbali-Fatourechi et al. (1996) Endocrinology 137(5): 1894-903). In the biomedical field, these types of assays are used to detect and characterize serum neutralizing antibodies against therapeutics, particularly protein therapeutics of which many are growth factors or cytokines.

The most widely used bioassay for serum neutralizing antibodies assesses cell proliferation by measuring the uptake of a radioisotope-labeled nucleotide, [³H]-thymidine (Eghbali-Fatourechi et al. (1996); Mire-Sluis (2001)). This approach can be used as long as the cells respond to the therapeutic agent by proliferating. By monitoring the amount of [³H]-Thymidine incorporated into chromosomes, either induction or inhibition of cell proliferation can be measured. When a neutralizing antibody is present, the therapeutic agent-induced proliferation is blocked. The major advantage of this method is its reliability and high sensitivity. The use of radioactive materials makes the method potentially hazardous and the disposal of radioactive waste increases the experimental costs, however. In addition, using cell proliferation as the final readouts results in a long assay duration time, typically ranging from 3 to 5 days.

Therefore, there is a need for reagents and safe, sensitive and effective methods for the detection of compounds that affect the activity of therapeutic substances and compositions.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence of a compound in a sample, comprising the following steps: providing, in any order: a sample suspected of comprising a compound and a control sample without the compound; a receptor and a response gene; and a ligand, wherein the ligand is capable of binding the receptor, thereby altering the expression of the response gene; combining, in any order, (i) the sample, the receptor, and the ligand; and (ii) the control sample, the receptor and the ligand; and measuring the level of the expression of the response gene; wherein the presence of the compound in the sample is detected by an alteration in the level of expression of the response gene when compared to the level of expression of the response gene when the receptor is combined with the ligand in the presence of the control sample. In one aspect, the invention provides methods for measuring the amount of a compound in a sample.

The invention further provides methods for detecting the presence of a compound in the presence or absence of a sample, comprising: providing, in any order: a compound, wherein the compound is in the presence or absence of a sample; a receptor and a response gene; and a ligand, wherein the ligand is capable of binding the receptor, thereby altering the expression of the response gene; combining, in any order, (i) the compound, the receptor, and the ligand; and (ii) the receptor and the ligand; and measuring the level of the expression of the response gene, wherein the presence of the compound is measured by an alteration in the level of expression of the response gene when the receptor is combined with the ligand and the compound compared to the level of expression of the response gene when the receptor is combined with the ligand only; and wherein when the receptor is combined with varying concentrations of the ligand and the compound, the expression of the response gene in the presence of the sample is correlated with the expression of the response gene in the absence of the sample with a correlation coefficient of at least 0.5. In one aspect, the method can be used for measuring the amount of the compound in the presence or absence of the sample.

In one aspect, the ligand can be a therapeutic substance for administration to a subject. In one aspect, the compound can be a neutralizing antibody against the therapeutic substance.

In one aspect, the receptor comprises SEQ ID NO:1. In another aspect, the receptor can comprise SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

In one aspect, the therapeutic substance comprises SEQ ID NO:6. In another aspect, the therapeutic substance can comprise SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In one aspect, the response gene can comprise SEQ ID NO:15. In another aspect, the response gene can comprise SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

In one aspect, the receptor can comprise the extracellular domain of SEQ ID NO:80. In another aspect, the receptor can comprise the extracellular domain of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83. In one aspect, the ligand can comprise SEQ ID NO:84. In another aspect, the ligand can comprise SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:91. In one aspect, the response gene can comprise SEQ ID NO:15.

In one aspect, the receptor can comprise the extracellular domain of SEQ ID NO:92. In another aspect, the receptor can comprise the extracellular domain of SEQ ID NO:93 or SEQ ID NO:94. In one aspect, the ligand can comprise SEQ ID NO:95. In another aspect, the ligand can comprise SEQ ID NO:96 or SEQ ID NO:97. In one aspect, the response gene can comprise SEQ ID NO:98. In another aspect, the response gene can comprise SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:103.

In one aspect, the response gene can comprise SEQ ID NO:15.

In one aspect, the ligand can comprise SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107. In one aspect, the receptor can comprise SEQ ID NO:108 or SEQ ID NO:109. In one aspect, the response gene is tartrate resistant acid phosphatase (TRAP).

In one aspect, the invention provides methods for detecting the presence of a compound in a sample or measuring the amount of a compound in a sample, wherein the ligand is an endogenous ligand, which is bound by a therapeutic substance for administration to a subject.

In one aspect, the level of the expression of the response gene is measured using a branched DNA (bDNA) assay.

In one aspect, the sample can be selected from the group consisting of whole blood, plasma, serum, synovial fluid, ascitic fluid, lacrimal fluid, perspiration, seminal fluid, cell extracts, and tissue extracts.

In one aspect, the invention provides methods for detecting the presence of a compound in a sample or measuring the amount of a compound in a sample, wherein the receptor is expressed by a mammalian cell.

In one aspect, the invention provides a kit comprising (a) a cell expressing a receptor, wherein the receptor comprises the intracellular domain of EPOR, and (b) one or more oligonucleotides used to detect PIM1 gene expression, the oligonucleotides selected from the group consisting of SEQ ID NOs: 22 through 79.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the EPO-induced PIM-1 expression in UT-7 cells. The level of PIM-1 mRNA expression in UT-7 cells treated with human rSCF, rG-CSF, rEPO, rGM-CSF, and mouse rIL-3 at indicated concentrations (A), or with 2 ng/mL of rEPO for indicated periods of times (B) was determined by using bDNA technology and compared with that in the untreated cells.

FIG. 2 schematically represents inhibition of EPO-induced PIM-1 expression by PI3-K antagonist. UT-7 cells pretreated with inhibitors for PI3-K, MAPK, PKA, and PKC and un-pretreated cells were treated with (+Epo) or without (−Epo) rEPO for 90 minutes (A) or 24 (B) hours. The levels of PIM-1 expression in cells treated with rEPO for 90 minutes were compared with that in untreated cells (A). The numbers of cells in cultures treated with or without rEPO for 24 hours were determined (B).

FIG. 3 illustrates EPO-induced PIM-1 expression in UT-7 cells in the presence of normal human serum. UT-7 cells were treated with 3 ng/mL of rEPO for 90 minutes in the presence or absence of indicated concentrations of normal human serum (A) or with rEPO at indicated concentrations for 90 minutes in the presence or absence of 10% normal human serum (B). The level of PIM-1 mRNA in each sample was determined and compared with that in untreated cells.

FIG. 4 is a schematic representation of inhibition of EPO-induced PIM-1 expression by Anti-EPO neutralizing antibody. UT-7 cells were treated with 0.6 ng/mL of rEPO with indicated concentrations of the anti-Epo neutralizing antibody in the presence or absence of 10% normal human serum. The expression level of PIM-1 in each sample was compared with that of untreated control cells.

FIG. 5 illustrates the detection of anti-EPO neutralizing antibodies in serum. UT-7 cells were treated with 10% of pooled (PNHS) or individual (D1-D10) normal human donor serum spiked with 200 or 400 ng/mL anti-EPO neutralizing antibody 29123 in the presence (purple, yellow, and orange bars) or absence (blue bars) of 0.6 ng/ml of rEpo at 37oC for 1.5 hours. The expression level of PIM-1 in each sample was determined by using bDNA technology. The cutoff line for assigning the presence of an anti-EPO neutralizing antibody (Emax/2) is calculated by (PIM-1 expression in cells treated with 10% pHS only + PIM-1 expression in cells treated with 10% pHS containing 0.6 ng/mL of rEPO)/2.

FIG. 6 schematically represents a comparison of gene expression and [3H]-Thymidine incorporation assay platforms. The levels of PIM-1 expression in UT-7 cells treated with indicated concentrations of rEPO (FIG. 6A), or with 0.6 ng/mL of rEPO and indicated concentrations of the anti-EPO antibody (FIG. 6B) for 1.5 hours were determined and compared with that in untreated control cells.

FIG. 7 is a schematic representation of NGF and EPO hybrid receptors.

FIG. 8 represents the generic cloning strategy for making NGFR/EPOR hybrid receptors.

FIG. 9 is a schematic representation of BAFFR and TNFR1 hybrid receptors.

FIG. 10 represents the generic cloning strategy for making BAFFR/TNFR1 hybrid receptors.

FIG. 11 is a schematic representation of IL-8 production (pg/ml) induced by BAFF in COS-1 cells transfected with BAFF/TNFR constructs.

FIG. 12 is a schematic representation of BAFFR and EPO hybrid receptors.

FIG. 13 represents the generic cloning strategy for making BAFFR/EPOR hybrid receptors.

FIG. 14 is a schematic representation of BAFF-induced PIM-1 expression in 32D cells expressing BMCEB constructs.

FIG. 15 is a schematic representation of the Nab Assay for RANK/RANK ligand in 29 human donors FIG. 16 represents the validation of the Specificity Value Threshold for RANK assay.

DETAILED DESCRIPTION OF THE INVENTION

I. Summary

The invention is directed to methods of detecting compounds that affect the activity of therapeutic substances or compositions, and to materials to be used in such methods. In one aspect, the method of the invention determines the activity of a therapeutic substance by measuring a cellular response to the binding between the therapeutic substance and a receptor for that substance, and comparing that response to the level of the response in the presence of a compound or compounds that may affect the binding between the therapeutic substance and its receptor. In other aspects, for example in cases where the therapeutic substance binds to an endogenous ligand and prevents or decreases the binding between the endogenous ligand and its receptor, the cellular response to binding of the receptor to its endogenous ligand is measured in the presence and absence of a compound that may affect the interaction of the therapeutic substance with the endogenous ligand.

II. Definitions “Polypeptide” is defined herein as natural, synthetic, and recombinant proteins or peptides generally having more than 10 amino acids. A “polypeptide linker” can be a polypeptide formed by a series of amino acids as short as one amino acid in length.

“Isolated”, as used herein, refers to a polypeptide or other molecule that has been removed from the environment in which it naturally occurs.

“Substantially purified”, as used herein, refers to a polypeptide that is substantially free of other polypeptides present in the environment in which it naturally occurs or in which it was produced; a preparation of a polypeptide that has been substantially purified contains at least 90% by weight (or at least 95%, at least 98%, or at least 99% by weight) of that polypeptide, wherein the weight of the polypeptide includes any carbohydrate, lipid, or other residues covalently attached to the polypeptide. A substantially purified polypeptide preparation may contain variation among polypeptide molecules within the preparation, with respect to extent and type of glycosylation or other post-translation modification, or with respect to conformation or extent of multimerization.

“Purified polypeptide”, as used herein, refers to an essentially homogenous polypeptide preparation; however, an essentially homogenous polypeptide preparation may contain variation among polypeptide molecules within the preparation, with respect to extent and type of glycosylation or other post-translation modification, or with respect to conformation or extent of multimerization.

“Full-length” polypeptides are those having the complete primary amino acid sequence of the polypeptide as initially translated; for example, the full-length form of the human EPO-R is shown as SEQ ID NO:2. The “mature form” of a polypeptide refers to a polypeptide that has undergone post-translational processing steps such as cleavage of the signal sequence and/or by proteolytic cleavage to remove a prodomain. Multiple mature forms of a particular full-length polypeptide may be produced, for example by cleavage of the signal sequence at multiple sites, or by differential regulation of proteases that cleave the polypeptide. The mature form(s) of such polypeptide can be obtained by expression, in a suitable mammalian cell or other host cell, of a nucleic acid molecule that encodes the full-length polypeptide. The sequence of the mature form of the polypeptide may also be determinable from the amino acid sequence of the full-length form, through identification of signal sequences or protease cleavage sites. In certain aspects, the mature form of the human EPO-R polypeptide has amino acid positions within the corresponding SEQ ID NOs as represented in Table 6.

The “percent identity” of two amino sequences can be determined by visual inspection and mathematical calculation, and the comparison can also be done by comparing sequence information using a computer program. The first step in determining percent identity is aligning the amino acid sequences to so as to maximize overlap and identities, while minimizing gaps in the alignment. The second step in determining percent identity is calculation of the number of identities between the aligned sequences, divided by the total number of amino acids in the alignment. When determining the percent identity that an amino acid sequence has “across the length of” a target amino acid sequence, the length of the target amino acid sequence is the minimum value for the number of total bases in the alignment. For example, when determining the percent identity of a first amino acid sequence of 50 amino acids “across the length of” a second amino acid sequence of amino acids 1 through 100 of SEQ ID NO:X, if the first amino acid sequence is identical to amino acids 1 through 50 of SEQ ID NO:X, the percent identity would be 50%: 50 amino acid identities divided by the total length of the alignment (100 amino acids). An exemplary computer program for aligning amino acid sequences and computing percent identity is the BLASTP program available for use via the National Library of Medicine website ncbi.nlm.nih.gov/gorf/wblast2.cgi, or the UW-BLAST 2.0 algorithm. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet site: sapiens.wustl.edu/blast/blast/README.html. In addition, the BLAST algorithm uses the BLOSUM62 amino acid scoring matrix, and optional parameters that can be used are as follows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton and Federhen (Computers and Chemistry, 1993); also see Wootton and Federhen, 1996, Analysis of compositionally biased regions in sequence databases, Methods Enzymol. 266: 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU program of Clayerie and States (Computers and Chemistry, 1993)), and (B) a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.); E-score threshold values are 0.5, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100. Other programs used by those skilled in the art of sequence comparison can also be used to align amino acid sequences, such as, the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, ‘GAP’ (Devereux et al., 1984, Nucl. Acids Res. 12: 387). The default parameters for the ‘GAP’ program include: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; or other comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps.

“Hybrid receptor” generally comprises an intracellular domain of one polypeptide joined to an extracellular domain of another polypeptide. The hybrid receptor further comprises a trans-membrane domain, which may be derived from either receptor or comprise a portion of one receptor and a portion of another one.

In one aspect, the extracellular domain includes amino acids 1-416, 1-417, 1-419, 1-422, or 1-424 of SEQ ID NO:81. In one aspect, the intracellular domain includes amino acid residues 274-507 of SEQ ID NO:3 In one aspect, the trans-membrane domain includes sequence GLAVFACLFLSTLLLVL.

In one aspect, the extracellular domain includes amino acids 1-68; 1-73 or 1-78 of SEQ ID NO:93. In one aspect, the intracellular domain includes amino acids 206-455 of SEQ ID NO:99. In one aspect, the trans-membrane domain includes sequence EDSGTTVLLPLVIFFGLCLLSLLFI.

In one aspect, the extracellular domain includes amino acids 1-68; 1-73 or 1-78 of SEQ ID NO:93. In one aspect, the intracellular domain includes amino acid residues 274-507 of SEQ ID NO:3. In one aspect, the trans-membrane domain includes sequence GLAVFACLFLSTLLLVL.

“Soluble forms” of polypeptides of the invention comprise certain fragments or domains of these polypeptides. Soluble polypeptides are polypeptides that are capable of being secreted from the cells in which they are expressed. A secreted soluble polypeptide can be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide. The presence of the desired polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the polypeptide. The use of soluble forms of cytokine polypeptides of the invention is advantageous for many applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Moreover, soluble polypeptides are generally more suitable than membrane-bound forms for parenteral administration and for many enzymatic procedures. In certain aspects of the invention, mature soluble forms of EPO-R or other polypeptides of the invention do not contain a trans-membrane or membrane-anchoring domain, or contain an insufficient portion of such a domain (e.g., 10 amino acids or fewer) to result in retention of the polypeptide in a membrane-bound form.

“An isolated polypeptide consisting essentially of an amino acid sequence” means that the polypeptide can optionally have, in addition to said amino acid sequence, additional material covalently linked to either or both ends of the polypeptide, said additional material between 1 and 10,000 additional amino acids covalently linked to either or both ends of the polypeptide; or between 1 and 1,000

additional amino acids covalently linked to either or both ends of the polypeptide; or between 1 and 100 additional amino acids covalently linked to either or both ends of the polypeptide. Covalent linkage of additional amino acids to either or both ends of the polypeptide according to the invention results in a combined amino acid sequence that is not naturally occurring.

“Correlation” and “correlation coefficient” are defined using the following formula: ${\rho }_{X,Y}=\frac{\mathrm{cov}\left(X,Y\right)}{{\sigma }_X·{\sigma }_Y}$ $\mathrm{where}$ ${\sigma }_X^2=1/n\sum {\left(X_i-{\mu }_X\right)}^2$ $\mathrm{and}$ ${\sigma }_Y^2=1/n\sum {\left(Y_i-{\mu }_Y\right)}^2$

This correlation formula measures the relationship between two data sets that are scaled to be independent of the unit of measurement. The population correlation calculation returns the covariance of two data sets divided by the product of their standard deviations. The correlation coefficient, symbolized in the formula above as ρx,y, will also be referred to herein using the symbol “r”. The correlation formula determines whether two ranges of data move together—that is, whether large values of one set are associated with large values of the other (positive correlation or positive value of r, with a maximum value of r=1), whether small values of one set are associated with large values of the other (negative correlation or negative value of r, with a minimum value of r=−1), or whether values in both sets are unrelated (correlation near zero, or value of r equal to or near zero).

III. Receptor Polypeptides for Detection of Neutralizing Antibodies

A. Summary. Receptor polypeptides of the invention are polypeptides that comprise at least a portion of the extracellular domain of a receptor polypeptide or of a variant thereof, covalently linked to at least a portion of the intracellular domain of a receptor polypeptide or of a variant thereof. The extracellular domain and the intracellular domain portions can be derived from same or from different receptor polypeptides, including embodiments wherein the extracellular portion of the receptor is from the human receptor, for example, and the intracellular portion of the receptor is from the murine form of the receptor.

Examples of different receptors, of ligands that interact with them, and of genes that are up- or down-regulated in response to receptor-ligand interactions, are provided in Tables 7-21 below.

B. Extracellular Domains. The receptor polypeptide for crystallization comprises at least a portion of the extracellular region of the polypeptide of SEQ ID NO:1, SEQ ID NO:80, SEQ ID NO:92, or SEQ ID NO:109, or a variant thereof. In certain aspects, the entire extracellular region of the polypeptide of SEQ ID NO:1, SEQ ID NO:80, SEQ ID NO:92, or SEQ ID NO:109 is included in the receptor polypeptide. As certain examples, the receptor polypeptide can comprise amino acids 25 through 251 of SEQ ID NO:1, or amino acids 33 through 420 of SEQ ID NO:80, or amino acids 1 through 79 of SEQ ID NO:9, or a variant thereof.

C. Intracellular Domains. The receptor polypeptide for crystallization comprises at least a portion of the intracellular region of the polypeptide of SEQ ID NO:1, SEQ ID NO:80, SEQ ID NO:92, or SEQ ID NO:109, or a variant thereof. In certain aspects, the entire intracellular region of the polypeptide of SEQ ID NO:1, SEQ ID NO:80, SEQ ID NO:92, or SEQ ID NO:109 is included in the receptor polypeptide. As certain examples, the receptor polypeptide can comprise amino acids 275 through 509 of SEQ ID NO:1, or amino acids 445 through 801 of SEQ ID NO:80, or amino acids 101 through 189 of SEQ ID NO:9, or a variant thereof.

D. Variants. Another consideration that will guide those of skill in the art in making variants of receptor polypeptides is the nature of the amino acid substitutions that are made; such substitutions can be conservative, which means that the amino acid present in the variant at a certain position has the same chemical and/or size properties as the amino acid at the corresponding position in the unaltered receptor polypeptide. Table 1 summarizes groups of amino acids that are considered to have similar properties, so that the substitution of any amino acid with another from the same row of Table 1 would be a conservative substitution. In certain aspects, receptor polypeptide variants have 20% or fewer amino acid substitutions (or 15% or fewer, or 10% or fewer, or 7.5% or fewer, or 5% or fewer, or 2.5% or fewer, or 1% or fewer) across the length of polypeptides of the invention. In certain aspects, receptor polypeptide variants have 20% or fewer conservative amino acid substitutions (or 15% or fewer, or 10% or fewer, or 7.5% or fewer, or 5% or fewer, or 2.5% or fewer, or 1% or fewer) across the length of polypeptides of the invention.

In certain embodiments, the receptor polypeptides or variants thereof have EPO-binding activity, NGF-binding activity, BAFF-binding activity, or RANKL (OPG)-binding activity.

TABLE 1
Conservative Amino Acid Substitutions
Basic:           arginine; lysine; histidine
Acidic:          glutamic acid; aspartic acid
Polar:           glutamine; asparagine
Alkyl:           leucine; isoleucine; valine; glycine;
                 alanine
Aromatic:        phenylalanine; tryptophan; tyrosine
Small Non-Alkyl: serine; threonine; cysteine; methionine;
                 proline

E. Expressing Receptor Polypeptides

The receptor polypeptides of the invention can be produced by living host cells that express the polypeptide, such as host cells that have been genetically engineered to produce the polypeptide. Methods of genetically engineering cells to produce polypeptides are well known in the art. See, e.g., Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, N.Y.). Such methods include introducing nucleic acids that encode and allow expression of the polypeptide into living host cells. These host cells can be bacterial cells, fungal cells, insect cells, or animal cells grown in culture. Bacterial host cells include, but are not limited to, Escherichia coli cells. Examples of suitable E. coli strains include: HB101, DH5α, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleave foreign DNA. Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Aspergillus cells. A few examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38. New animal cell lines can be established using methods well known by those skilled in the art (e.g., by transformation, viral infection, and/or selection).

Purification of the expressed receptor polypeptide can be performed by any standard method. When the receptor polypeptide is produced intracellularly, the particulate debris is removed, for example, by centrifugation or ultrafiltration. When the polypeptide is secreted into the medium, supernatants from such expression systems can be first concentrated using standard polypeptide concentration filters. Protease inhibitors can also be added to inhibit proteolysis and antibiotics can be included to prevent the growth of microorganisms. Receptor polypeptides can be produced in the presence of chaperone or accessory proteins in order to obtain a desired polypeptide conformation, or can be subjected to conditions such as oxidizing and/or reducing conditions after production in order to induce refolding or changes in polypeptide conformation (see, for example, WO 02/068455).

The receptor polypeptide can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and any combination of purification techniques known or yet to discovered.

F. Gene Expression Methods

The invention provides methods of detecting compounds that affect therapeutic activity by measuring gene expression of response genes.

In one aspect, the expression of PIM-1, a protein serine/threonine kinase potentially involved in EPO-dependent survival and proliferation of erythroid precursors and other types of cells (Meeker et al. (1987) J. Cell. Biochem. 35: 105-12.; Wang et al., (2001) J Veterinary Sci. 2(3): 167-79; Kumenacker et al. (2001) J. Neuroimmunology 133: 249-259), and regulated by EPO in EPO-dependent UT-7 cells, can be used for detecting compounds affecting therapeutic activity of EPO. Previous studies have shown that PI3-K activity is critical for EPO-supported erythroid progenitor survival and differentiation (Kumenacker et al. (2001); Myklebust et al. (2002) Exp. Hematol. 30:990-1000; Uddin et al. (2000) Biochem. Biophys. Res. Comm. 275: 16-9; Sawyer and Jacobs-Helber, (2000) J Hematotherapy & Stem Cell Research 9:2 1-9). Data presented in Example 1 indicate that this signal is also essential for the EPO-dependent survival and/or proliferation of the UT-7 cells. Although the exact function of PIM-1 in EPO-stimulated cell survival and proliferation remains unclear, these observations suggest that it may be a signal transducer playing roles down stream of the PI3-K. At least, the expression of PIM-1 itself in EPO-dependent UT-7 cells is clearly coregulated by PI3-K signaling. Therefore, the level of PIM-1 mRNA expressed in EPO-dependent UT-7 cells reflects the amount of EPO signal received by the cells and can be used as a quantitative measurement for EPO signaling. Thus, the same strategy can be used for determining the presence and quantitative measurement of anti-EPO neutralizing antibodies that inhibit the biological activities of EPO.

In another aspect, PIM-1 expression can be used for detection of compounds, for example, neutralizing antibodies, that affect activity of therapeutic protein or antibodies/peptibodies by generating hybrid constructs using, for example, an intracellular domain of the EPO receptor linked to an extracellular domain of the receptor of interest. In one aspect, an intracellular domain of NGF receptor can be used. In another aspect, BAFF receptor can be used.

In another aspect, different response genes can be used. In one aspect, IL-8 expression can be used as a quantitative measurement. Thus, a construct using, for example, an intracellular domain of a TNF receptor and an extracellular domain of a receptor of interest can be created. In one aspect, a hybrid construct comprising the extracellular domain of BAFFR and the intracellular domain of TNFR can be created and thereby BAFF-induced IL-8 expression can be used to detect anti-BAFF neutralizing antibody, for example.

In yet another aspect, mRNA expression of the terminal differentiation marker TRAP (tartrate-resistant acid phosphatase) can be used (Lacey et al. (1988)). The inhibition of OPG ligand/RANK by antibodies or peptibodies would inhibit TRAP production as well, however, if compounds affecting anti-RANK antibodies were present, such as neutralizing antibodies, the TRAP enzyme would continue to be produced.

The above description of the invention is exemplary, and is not meant to be limiting as to, for example, the response gene, the types of host cells used, the methods and compositions for providing for host cells having varying levels of expression of a response gene, and the like, as such can be varied and remain within scope of the present invention, and such variations will be readily appreciated by the ordinarily skilled artisan.

G. Branched DNA Technology Expression of a response gene can be detected in a variety of ways well known in the art, e.g., by use of hybridization probes, PCR primers, or antibodies specific for a response gene product.

In one aspect, branched DNA (bDNA technology) can be used to quantitatively measure expression of a response gene. This is a highly sensitive and convenient to use technique. Urdea, M. and Wuestehube, L. (2000). Branched DNA (bDNA) technology. In: C. Kessler (Ed.) Nonradioactive Analysis of Biomolecules. Srpinger-Verlag Press & Publications, Heidelberg, p. 388. It can detect the existence of as few as 1 to 50 copies of mRNA in a sample. Elbeik, T. et al. (2000) J. Clin. Microbiol. 38: 1113-20. The entire procedure can be fully automated, making it a good choice for high throughput operations. Murphy, D. G. et al. (2000) J. Clin. Microbiol. 38: 4034-41.

The above description of the invention is exemplary, and is not meant to be limiting as to, for example, the methods for detecting mRNA of the response gene, and the like, as such can be varied and remain within scope of the present invention, and such variations will be readily appreciated by the ordinarily skilled artisan.

For example, rather than detect changes in expression of a response gene by using bDNA technology, response gene expression can be detected by using methods well known in the art for detecting gene expression levels (e.g., Northern blot, microarray-based, or other solid support-based methods, tailored expression membrane assays, or Taqman assays etc.). Also it will be readily appreciated that increasing the number of response genes analyzed (e.g., two or more response genes) can provide additional information and/or increase confidence scores of the results obtained relative to detection of a single response gene. Assays with multiple response genes can be conducted simultaneously using different detectable labels for each gene (e.g., different fluorescent reporters having different excitation and/or emission wavelengths).

H. Introduction into Host Cells

Methods for introducing a construct into a host cell are well known in the art. This can be accomplished by, for example, introduction of an autonomous plasmid, which can be maintained as an episomal element and/or chromosomally integrated into the genome of the host cell. Suitable constructs, vectors, plasmids, etc. are well known in the art and will vary with the host cell, size and other characteristics of the reporter gene, etc.

The methods of the invention can be used in connection with any of a variety of host cells, including eukaryotic, prokaryotic, diploid, or haploid organisms. Host cells can be single cell organisms (e.g., bacteria) or multicellular organisms (transgenic organisms, such as insects (e.g., Drosophila spp), worms (e.g., Caenorhabditis spp, e.g., C. elegans) and higher animals (e.g., transgenic mammals such as mice, rats, rabbits, hamsters, humans etc. or cells isolated from such higher animals, including humans). The host cell can also be a cell infected with a virus or phage that contains a target sequence in the viral or phage genome.

The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1

This example illustrates an assay which measures the variations of target gene expression that reflect the biologic effect of a therapeutic agent and capabilities of the antibodies, if present, to neutralize the therapeutics. In particular, this method can be used for detection and measurement of anti-erythropoietin antibodies.

Cells and Proteins

UT-7, a human acute megakaryocytic leukemia cell line was maintained in growth media [RPMI/1640 (Gibco, N.Y.) containing 10% fetal calf serum (Hyclone, Logon, Utah)] supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). Recombinant human erythropoietin (rEPO), stem cell factor (rSCF), granulocyte colony-stimulating factor (rG-CSF), rGM-CSF, mouse interleukin-3 (rIL-3), and rabbit anti-human EPO polyclonal antibody (29123) were provided by Amgen. Protein kinase inhibitors for phosphatidylinositol 3-kinase (PI3-K) (LY294002), MAP kinase (MAPK) (UO126), protein kinase A (PKA), and protein kinase C (PKC) were all purchased from Promega (Madison, Wis.).

Microarray Analysis of Gene Expression

Microarray analysis of cell samples was performed using well-described protocols (Eisen and Brown (1999) Methods Enzymol. 303, 179-205) with minor modifications. PolyATtract (Promega) purified mRNA was reverse-transcribed using random primers in the presence of either Cy3 or Cy5 dye-labeled dCTP. Control and test fluorescent probes were hybridized to cDNA-spotted glass slides overnight in a competitive hybridization process. After washing, fluorescent images of the dried slides were obtained using a GenePix Scanner 4000 (Axon Instruments, Union City, Calif.). GenePix Pro 3.0 software (Axon Instruments, Union City, Calif.) was used for feature detection. The subsequent data were inspected using a series of internal standards that enabled determination of sensitivity, linearity, and dynamic range of response within individual experiments. Global normalized data was then exported into the Resolver (Rosetta, Kirkland, Wash.) database for storage and analysis. For each control and test pair, data was combined with a second dye-swapped hybridization to reduce potential dye incorporation biased measurements.

Oligonucleotide Probes

Human PIM-1 specific probes and human cyclophilin probes for bDNA analysis were designed by using the ProbeDesigner software from Bayer Corporation (West Haven, Conn.). Three sets of oligonucleotide probes were designed for each molecule: the capture extender (CE), label extender (LE), and blocker (BL). Thirty-one probes were generated for PIM-1, including 8 CE probes, 17 LE probes, and 6 BL probes; and 27 were made for cyclophilin, including 6 CE probes, 18 LE probes, and 3 BL probes (Table 2). All probes for each gene were pooled according to the manufacturer's instructions.

TABLE 2
Oligonucleotide probes for bDNA detection
   Human PIM-1
CE gctcgggcctctccaggTTTTTCTCTTGGAAAGAAAGT
   agtcgaagagatcttgcaccgTTTTTCTCTTGGAAAGAAAGT
   ggccagctcctcttgcaggTTTTTCTCTTGGAAAGAAAGT
   agctcgccgcgattgagTTTTTCTCTTGGAAAGAAAGT
   ttgagcagcgcccccgaTTTTTCTCTTGGAAAGAAAGT
   catacagcaggatccccaggTTTTTCTCTTGGAAAGAAAGT
   attctgaagagaccctctgcctgaaTTTTTCTCTTGGAAAGAAAGT
   gatttcttcgaaggttggcctTTTTTCTCTTGGAAAGAAAGT
LE gcaggaccacttccatgggTTTTTAGGCATAGGACCCGTGTCT
   acccgagctcaccttcttcaTTTTTAGGCATAGGACCCGTGTCT
   gcctaatgacgccggagaaTTTTTAGGCATAGGACCCGTGTCT
   cctctcgaaccagtccaggaTTTTTAGGCATAGGACCCGTGTCT
   atcaggacgaaactgtcgggTTTTTAGGCATAGGACCCGTGTCT
   gctcccctttccgtgatgaTTTTTAGGCATAGGACCCGTGTCT
   gccgcacggcctccagTTTTTAGGCATAGGACCCGTGTCT
   ccccgcagttgtggcagtTTTTTAGGCATAGGACCCGTGTCT
   tgatgtcgcggtgtagcaTTTTTAGGCATAGGACCCGTGTCT
   gtcgataaggatgttttcgtcctTTTTTAGGCATAGGACCCGTGTCT
   aagtccgtgtagacggtgtccTTTTTAGGCATAGGACCCGTGTCT
   ctatacactcgggtcccatcgTTTTTAGGCATAGGACCCGTGTCT
   ctgccatggtagcgatggtaTTTTTAGGCATAGGACCCGTGTCT
   gaccagactgccgccgacTTTTTAGGCATAGGACCCGTGTCT
   aaggaatatctccacacaccatatTTTTTAGGCATAGGACCCGTGTC
   T
   agcaccatctaatgagatgctgacTTTTTAGGCATAGGACCCGTGTC
   T
   ttgcatccatggatggttctgTTTTTAGGCATAGGACCCGTGTCT
BL cacctgccagaagaagctgcg
   cccgaagtcgatgagcttg
   gcggatccactctggaggg
   gatctcttcgtcatgctcga
   gaaaacctggcccctgat
   atctgatggtctcagggcca
   Human Cyclophilin
CE atacaccttgacggtgactttgTTTTTCTCTTGGAAAGAAAGT
   cctttctctcctgtagctaaggcTTTTTCTCTTGGAAAGAAAGT
   ggtgtctttgcctgcgttgTTTTTCTCTTGGAAAGAAAGT
   tgaagaactgggagccgttTTTTTCTCTTGGAAAGAAAGT
   gtctgtcttggtgctctccaTTTTTCTCTTGGAAAGAAAGT
   tcaggggtttatcccggctTTTTTCTCTTGGAAAGAAAGT
LE gccgcagaaggtcccggcTTTTTAGGCATAGGACCCGTGTCT
   ggccccttcttcttctcatcgTTTTTAGGCATAGGACCCGTGTCT
   catctccaattcgtaggtcaaaTTTTTAGGCATAGGACCCGTGTCT
   agatcacccggcctacatcttTTTTTAGGCATAGGACCCGTGTCT
   cacaaaattatccactgtttttggTTTTTAGGCATAGGACCCGTGTC
   T
   atttgctgtttttgtagccaaatTTTTTAGGCATAGGACCCGTGTCT
   agtctccgccctggatcatTTTTTAGGCATAGGACCCGTGTCT
   tgtgccatctcccctggtgaTTTTTAGGCATAGGACCCGTGTCT
   accgtagatgctctttcctccTTTTTAGGCATAGGACCCGTGTCT
   agttctcatcggggaagcgctTTTTTAGGCATAGGACCCGTGTCT
   aggcccgtagtgcttcagtttgaTTTTTAGGCATAGGACCCGTGTCT
   gccatgctgacccagccTTTTTAGGCATAGGACCCGTGTCT
   acatgcttgccatctagccaTTTTTAGGCATAGGACCCGTGTCT
   ccctctagaactttgccaaacaccTTTTTAGGCATAGGACCCGTGTC
   T
   ccttccgcaccacctccatgTTTTTAGGCATAGGACCCGTGTCT
   cagtctgcgatgatcacatcctTTTTTAGGCATAGGACCCGTGTCT
   tccacctcgatcttgccgTTTTTAGGCATAGGACCCGTGTCT
   gcgatggcaaagggcttcTTTTTAGGCATAGGACCCGTGTCT
BL aacagtctttccgaagagaccaa
   gaagtccttgattacacgatgga
   ggctgtcttgactgtcgtga

Cell Treatment

UT-7 cells were washed 2 times with the growth media and incubated overnight in rGM-CSF-free media at 37° C. with 5% CO₂. Triplicate samples of the rGM-CSF-starved cells were seeded in 96-well tissue culture plates with 100 μL rGM-CSF-free media at a density of 1.2×10⁵ cells per well and treated with various concentrations of rEPO in the absence or presence of anti-EPO antibody, or with various concentrations of other cytokines, including rGM-CSF, rG-CSF, rSCF, and rIL-3, at 37° C. for 90 minutes.

For serum tolerate experiments, GM-CSF-starved UT-7 cells were either treated with 3 ng/mL rEPO in the presence or absence of various concentrations pooled normal human serum (PNHS, Bioreclamation, Inc., East Meadow, N.Y.) or with various concentrations of rEPO in the presence or absence of 10% PNHS at 37° C. for 90 minutes.

For studies using signal transduction antagonists, the rGM-CSF-starved cells were first treated with various concentrations of LY294002, UO126, and the inhibitors for PKA and PKC separately at 37° C. for 30 minutes and then with 21 ng/mL of rEPO at 37° C. for 90 minutes or 24 hours. After all treatments, the levels of PIM-1 expression were determined using branched DNA (bDNA) technology. The number of cells in each well that had been treated with rEPO for 24 hours was counted.

Branched DNA Analysis

Branched DNA analysis was performed using the QuantiGene High Volume Kit (Bayer, West Heaven, Conn.) using a 3-step procedure provided by the manufacturer, which included specimen preparation, hybridization, and detection. Briefly, treated or untreated UT-7 cells seeded in 96-well tissue culture plates were mixed with 50 μL lysis mixture (provided by the kit) using a multiple channel pipette and incubated at 46° C. for 30 minutes to release mRNA. Aliquots of 70 μL and 30 μL of each lysate were transferred to capture plates (provided by the kit) with 30 μL pooled PIM-1-specific probes or 70 μL pooled cyclophilin probes, respectively, and incubated overnight at 53° C. The hybridization mixtures were removed and the plates were washed twice with 400 μL wash buffer (0.1 SSC, 0.03% lithium lauryl sulfate) using an Auto Plate Washer (Bio-TEK, Winooski, Vt.). After washing, 100 μL Amplifier Working Reagent (provided by the kit) was added to each well and incubated at 46° C. for 1 hour. The plates were washed twice as described above, incubated with 100 μL Labeling Working Reagent (provided by the kit) at 46° C. for 1 hour, washed again 3 times, and processed for chemiluminescent detection. The amount of the target mRNA in each sample was determined by the intensity of the luminescent emission detected using a luminometer (Wallac Victor 1420, Perkin Elmer, Finland).

[³H]-Thymidine Incorporation

UT-7 cells were washed 2 times with growth media and incubated overnight in rGM-CSF-free media at 37° C., with 5% CO₂ as described above. Triplicate samples of the rGM-CSF-starved cells were seeded in 96-well tissue culture plate at a density of 1×10⁵ cells per well and treated with various concentrations of rEPO in the presence or absence of anti-EPO antibody at 37° C. with 5% CO₂ for 72 hours. Then, 2 μCi [³H]-thymidine (Amersham, Little Chalfont, Buckinghamshire, UK) were added to each well and the cells were further incubated for 4 hours. The cells were harvested using a cell harvester (Filtermate 196, Packard, Ill.), and the incorporated radioactivity was determined using a Matrix 9600 beta counter (Packard, Ill.).

EPO Induces PIM-1 Expression

mRNA microarray experiments determined which genes in UT-7 cells had altered expression after rEPO treatment. UT-7 cells quieted in rGM-CSF-free media were treated with 20 ng/mL rEPO at 37° C. for 2, 4, 6, or 24 hours. Messenger RNAs extracted from the rEPO-treated or untreated control cells were used to generate probes for the subsequent microarray experiments. The mRNA expression level of a number of genes has been changed in rEPO-treated cells compared with that in the untreated control cells (Table 3). In particular, the level of the PIM-1 mRNA in rEPO-treated cells was more than 20 times higher than that in the untreated control cells.

TABLE 3
Epo-Altered mRNA expression in UT-7 cells
	Folds*
	Gene Name	2 hr	4 hr	6 hr	24 hr
	PIM 1	+8.06	+13.85	+8.49	+21
	LOC64182	+2.96	+6.42	+3.98	+13.68
	RTP801	+2.47	+3.75	+3.68	+9.64
	NFYC	+1.48	+2.07	+2.39	+8.25
	CBS	+1.43	+1.47	+1.66	+7.59
	IGFBP4	+2.14	+2.91	+2.82	+5.57
	ATF5	+1.12	+1.53	+1.67	+5.22
	CTH	+1.32	+1.97	+2.66	+5.16
	PKM2	+1.30	+1.43	+1.52	+5.15
	LDHA	+1.31	+1.83	+2.24	+4.80
	JTB	+1.42	+1.82	+2.53	+4.33
	GPI	+1.32	+1.47	+1.50	+4.24
	VEGF	+1.88	+2.05	+2.32	+3.81
	ATF4	+1.85	+1.95	+2.39	+3.60
	LDHC	+1.35	+1.63	+2.41	+3.31
	TIMP1	+1.50	+1.50	+1.60	+3.16
	POLD1	−2.45	−1.35	+1.13	−2.18
	GCH1	−2.81	−1.14	+1.08	−2.21
	EIF4G3	−1.25	−1.33	−1.40	−2.48
	SPTA1	−1.83	−1.77	−1.74	−2.64
	RYR3	−1.79	−1.91	−1.80	−2.83
	MGLL	−2.77	−1.12	1.09	−284
	AKR1C3	−1.01	−1.08	−1.13	−2.92
	*Represent the ratio of the mRNA levels in EPO-treated cells versus that in the untreated cells

The EPO upregulated PIM-1 mRNA expression in UT-7 cells was further confirmed using bDNA technology (FIG. 1A). Similar to rEPO, rGM-CSF also induced expression of the PIM-1 mRNA in UT-7 cells (FIG. 1A), while rSCF, rG-CSF, and IL-3 showed no effect. The magnitude of the rEPO-induced PIM-1 expression in UT-7 cells was dose dependent and the maximum induction was reached after 90 minutes of rEPO treatment (FIG. 1B).

PIM-1 Expression is Regulated by PI3K Signaling

Chemical antagonists of the major intracellular signal transduction molecules downstream of the EPO receptor were used to determine which of the EPO signaling pathways were involved in the up-regulated PIM-1 expression. LY294002, a PI3-K inhibitor, effectively inhibited EPO-induced PIM-1 expression after 30 minutes of pre-incubation and the inhibition was apparently in a dose-dependent manner (FIG. 2A). All other antagonists showed either no or very mild effects on the regulated PIM-1 expression. When cells were treated for 24 hours, LY294002 blocked EPO-induced UT-7 cell proliferation and appeared to have activated apoptosis of the cells (FIG. 2B).

Effects of Serum on EPO-Induced PIM-1 Expression

To determine if PIM-1 expression could be used as a biological measurement for detecting neutralizing antibodies in serum samples, the effect of serum concentrations on EPO-induced PIM-1 expression were evaluated. As shown in FIG. 3, up to 20% of PNHS was well tolerated by this assay system.

Detection of Anti-EPO Neutralizing Antibodies in Serum

The rabbit polyclonal antibody 29123 is an EPO specific neutralizing antibody that has been shown to inhibit the survival and proliferation of UT-7 cells in media supplemented with rEPO. In these experiments, this antibody effectively inhibited EPO-induced elevation of PIM-1 expression (FIG. 4). The inhibition was dose dependent and was not affected by the presence of 10% normal human serum. Apparent inhibition could be observed at 2 ng/mL of 29123, and the maximum effect was reached at approximately 30 ng/mL.

The feasibility of using PIM-1 expression as a biological measurement for detecting anti-EPO neutralizing antibodies in serum samples was further evaluated by a more realistic spiking experiment. The levels of PIM-1 expression in cells treated with each donor serum only were compared with expression levels of the cells treated with the serum plus rEPO or rEPO and the spiked antibody. When 400 ng/mL antibody was spiked into the donor sera, EPO-induced PIM-1 expression was almost completely blocked by all of the spiked samples (FIG. 5). When 200 ng/mL antibody was spiked, however, effective, but weaker inhibition was observed. In either case, the levels of PIM-1 expressed in all antibody-treated samples were well below the tentative cutoff line for positive assignment. Additionally, the result indicated once again that PIM-1 expression was not influenced by the presence of at lease 10% of human serum, implying that a PIM-1 expression-based assay system could tolerate assay matrixes with high concentrations of serum for potential improvement of assay sensitivities.

Gene Expression Vs [³H]-Thymidine Incorporation

As shown in FIG. 6A, the pattern of EPO-induced PIM-1 expression was almost superimposable to that of EPO-induced [³H]-thymidine incorporation in UT-7 cells. The Pearson's correlation coefficient between the increased PIM-1 expression and [³H]-thymidine incorporation was 0.974, confirming that these 2 events were highly related. The maximum induction and the EC₅₀ for EPO-induced PIM-1 expression were approximately 22-fold and 0.4 ng/mL, while the same for [³H]-thymidine incorporation were approximatelyl 9-fold and 1 ng/mL. The blocking of EPO-induced PIM-1 expression and that of [³H]-thymidine incorporation by anti-EPO antibodies were also highly related (FIG. 6B). The Pearson's correlation coefficient between these 2 processes was 0.958. The maximum inhibition of PIM-1 expression and [³H]-thymidine incorporation were approximately 11-fold and 6-fold; while the IC₅₀ for PIM-1 expression and [³H]-thymidine incorporation were approximately 3.4 ng/mL and 5.4 ng/mL, respectively (FIG. 6). These numbers indicated that the gene expression approach was more robust and sensitive than the [³H]-Thymidine incorporation method.

EXAMPLE 2

This example illustrates the application of the method to NGFR (Nerve growth factor receptor) and EPOR hybrid receptors. Briefly, five different human NGFR and EPOR hybrid receptors (NECA-NECE, NGFR/EPOR chimera A-E) have been constructed. Different lengths of extracellular domain of human NGFR were fused with the mouse EPO receptor trans-membrane and intracellular domains (FIG. 7). The amino acid sequence at the bottom of FIG. 7 represents the junction points of hybrid receptors, wherein the trans-membrane domain is italicized and the 3′ end of NGFR extracellular sequence is underlined. The black bars indicate the sequence position where the 3′-end of the NGF was fused to EPOR in each chimeric construct. FIG. 8 represents a generic cloning strategy for making NGFR/EPOR hybrid receptors. Human NGF receptor extracellular domain fragment was obtained by PCR followed by restriction enzyme digestion by Not I at 5′ end and Spe I at 3′ end. A fragment containing mouse EPOR trans-membrane and intracellular domains was obtained by PCR followed by digestion by Spe I at 5′ end and Sal at 3′ end. Two fragments were ligated and subcloned into the pLJ vector cut with Not I and Sal I. Positive clones were obtained and sequenced to confirm sequence.

These five different forms of NGFR/EPOR hybrid receptor constructs were transfected into 32Dcl3 cell via electroporation and were selected by medium containing G418 and NGF to yield 32D/NECD cells. A NGF responsive cell line NECDsc-14 was generated after two rounds of selection and single cell subcloning. These 32D/NECDsc-14 cells were maintained in either 5 ng/ml mouse interleukin-3 (mIL-3) or 25 ng/ml of NGF. NGF induced NECDsc-14 cell proliferation can be measured by [3H]-uptake. Cells were washed three times, staged overnight with growth factor-free culture medium, and treated with various amount of mIL-3 (control) or NGF (as indicated in Table 4) for 18 hours. The amounts of [³H]-thymidine incorporated into the cells were measured following further incubation of the cells with [³H]-thymidine for 4 hours. Results of this experiment are represented in Table 4 below.

TABLE 4
NGF Dose Response
Counts
Conc	NECDsc-14	NECDsc-14
(ng/ml)	mIL-3-dependent	NGF-dependent
0.00	655.3	1209.3
0.02	1724.7	1963.3
0.07	3784.0	4691.0
0.21	8645.7	8033.0
0.62	18964.0	16838.0
1.85	28205.7	22404.0
5.56	32071.3	24205.3
16.67	31791.3	22907.0
50.00	30813.7	21618.3
150.00	32221.7	20857.3

This NGF induced proliferation of NECDsc-14 cells can be used to detect anti-NGF neutralizing antibodies or peptibodies, which inhibit the NGF-induced proliferation of NECDsc-14 cells, in biological samples similar to the assay described in Example 1. Similarly, it can be used to detect neutralizing antibodies against the anti-NGF antibodies or peptibodies, which reverse the inhibitory effects mentioned above, in biological samples. Assays for detecting and measuring the concentrations of neutralizing antibodies against anti-NGF antibodies or peptibodies can be performed in 1% human serum, 5% cynomolgus monkey serum, or 2% rat serum samples (see Table 5), as no significant matrix effect from these samples was observed.

TABLE 5
NGF Dose Response in Serum Matrix
NGF conc.		5% cyno	2% rat	1% human
(ng/mL)	No serum	serum	serum	serum
0.00	462.7	1534.7	702.7	367.3
0.02	595.3	2175.7	1188.3	946.7
0.07	1389.7	5761.7	3524.0	1480.0
0.21	5423.0	15774.0	11970.7	5832.3
0.62	17073.0	32462.7	29431.7	13374.7
1.85	27445.3	43119.7	37695.3	19493.7
5.56	35224.7	52240.7	43395.7	22160.0
16.67	39675.3	51793.3	42446.0	24558.7
50.00	41018.0	56389.7	46874.3	19712.0
150.00	44119.0	62140.0	49545.3	18626.7

EXAMPLE 3

This example illustrates the application of the method to detecting the presence and measuring the concentration of neutralizing antibodies against an anti-BAFF antibody or peptibody. In particular, it is demonstrated that a BAFF-induced release of IL-8 (interleukin-8) that can be measured by ELISA can serve to identify and measure the cellular response to BAFF (B cell Activating Factor).

To measure the release of IL-8, a BAFF/TNFR hybrid receptor was constructed. As demonstrated in FIG. 9, three different human BAFFR and human TNFR hybrid receptors (B1T-B3T, BAFFR/TNFR hybrid receptors 1-3) were constructed. Extracellular domains of the human BAFF receptor of different length (BMCA, 68 amino acids; BMCB, 73 amino acids; BMCC, 78 amino acids) were fused with the human TNF receptor domain consisting of amino acids 206-455. The amino acid sequence in FIG. 9 represents the junction point of the hybrid receptor, wherein the trans-membrane domain area of the TNF is italicized and the 3′ end of the BAFFR extracellular domain is underlined.

FIG. 10 outlines a generic cloning strategy for making BAFF/TNFR hybrid receptors. Briefly, a fragment of an extracellular domain of the human BAFFR was obtained by PCR followed by restriction enzyme digestion by Not I at the 5′ end and Nhe I at the 3′ end. A fragment including the trans-membrane and the intracellular domains of the human TNFR was obtained by PCR and restriction enzyme digestion by Xba I at 5′ end and Xho I at the 3′ end. Two fragments were ligated and subcloned into the pCEP4 vector cut with Not I and Xho I. Positive clones were obtained and sequenced to confirm sequence.

COS-1 cells were stably transfected with the BAFFR/TNFR chimeric constructs and selected in Hygromycin to yield clones of the hybrid receptor expressing COS-1 cells. The final clones of the cells expressing the BAFF/TNFR chimera were tested in a BAFF induced IL-8 release from COS-1 cells expressing BAFFR/TNFR hybrid. Briefly, COS-1 cells were transfected stably with the BAFFR/TNFR chimeric construct (B2T) and were selected in Hygromycin to yield COS-1/B2T cells. FIG. 11 illustrates the IL-8 production (in pg/ml) by cell lines PCEP4, B1T-3, B2T-17, and B2T-20 FIG. 11 illustrates the IL-8 production (in pg/ml) by cell lines PCEP4, B1T-3, B2T-17, and B2T-20. The cells were then seeded in a 12-well plate and treated with BAFF at indicated concentrations at 37^oC for 18 hours. The conditioned medium was collected and the IL-8 production was measured by ELISA kit (R&D systems). Because of the highest IL-8 production, B2T-17 line was selected for the rest of the study.

This BAFF-induced IL-8 expression from B2T-17 cells can be used in biological sample to detect anti-BAFF neutralizing antibody or peptibody, which inhibits the BAFF-induced IL-8 release from B2T-17 cells. Similar to he assay described in Example 1, the same construct can be used to detect neutralizing antibodies against the anti-BAFF antibodies or peptibodies, which reverse the inhibitory effects mentioned above. Assays for detecting and measuring the amount of neutralizing antibodies against anti-BAFF antibodies or peptibodies can be performed in 1% human or 5% cynomolgus monkey serum, as no significant matrix effect from these samples was observed.

EXAMPLE 4

This example illustrates the application of the method to detecting the presence and measuring the concentration of neutralizing antibodies against an anti-BAFF antibody or peptibody by measuring an alteration in the BAFF-induced PIM-1 expression.

A mouse EPOR and human BAFFR hybrid receptor was constructed as represented in FIG. 12, wherein the EPOR trans-membrane domain is indicated in black. Briefly, three different human BAFFR and mEPOR hybrid receptors (BMCA-C) have been constructed. Extracellular domains of human BAFFR (of three different length: BMCA, 68 amino acids; BMCB, 73 amino acids; BMCC, 78 amino acids) were fused with the mouse EPO receptor comprising the trans-membrane and the intracellular domain. The amino acid sequence representing the junction point of the hybrid receptor is shown in FIG. 12, wherein the trans-membrane domain of the mouse EPOR is italicized and the 3′ end of the human BAFFR extracellular sequence is underlined.

FIG. 13 outlines a generic cloning strategy for making BAFFR/EPOR hybrid receptors. A fragment containing the extracellular domain of human BAFF receptor was obtained by PCR and restriction enzyme digestion by Not I at the 5′ end and Nhe I at the 3′ end. A fragment containing the mouse EPOR trans-membrane and intracellular domain was obtained by PCR and restriction enzyme digestion by Spe I at the 5′ end and Sal at the 3′ end. Two fragments were ligated and subcloned into pLJ vector cut with Not I and Sal I. Positive clones were obtained and sequenced to confirm sequence.

These three different forms of BAFFR/EPOR hybrid receptor construct were transfected into 32Dcl3 cell via electroporation and were selected by medium containing G418 and BAFF to yield 32D/BMC cells. A BAFF responsive cell line BMECB was generated after two rounds of selection and single cell subcloning. These 32D/BMECB cells were maintained in either 5 ng/ml mouse interleukin-3 (mIL-3) or 25 ng/ml of BAFF. BAFF induced PIM-1 expression from 32D/BMECB cells can be measured by bDNA technology. Briefly, three subclones of BMECB cells (BMECB-9, 20, 21) were washed three times, staged overnight with growth factor-free culture medium. Cells were then seeded in 96-well plate in triplicate and treated with BAFF at indicated concentrations at 37° C. for 90 minutes as represented in FIG. 14. Treated cells were lysed and the amount of PIM-1 expression induced by the BAFF treatment was measured by using bDNA QuantiGene kit and illustrated in FIG. 14.

This BAFF-induced expression of PIM-1 in 32D/BMECB cells can be used in biological samples to detect anti-BAFF neutralizing antibody or peptibody, which inhibit the BAFF-induced expression of PIM-1 in 32D/BMECB cells. Similarly, it can be used to detect neutralizing antibodies against the anti-BAFF antibodies or peptibodies, which reverse the inhibitory effects mentioned above. Assays for detecting and measuring the concentrations of neutralizing antibodies against anti-BAFF peptibodies can be performed in 1% human serum, 5% cynomolgus monkey serum, or 2% rat serum samples (Table 5), as no significant matrix effect from these samples was observed.

EXAMPLE 5

This Example illustrates the validation of a cell-based Neutralizing Antibody (NAb) bioassay for the detection of specific neutralizing activity to a therapeutic protein, such as Osteoprotegerin (OPG) or a monoclonal anti-Receptor Activator of NFκB (RANK) ligand antibody (anti-RANKL) in human serum measuring changes in TRAP (tartrate-resistant acid phosphatase) mRNA using Branched DNA (bDNA) technology (Quantigene, Genospectra, Inc. Fremont, Calif.).

For the determination of neutralizing effects of a human Fc conjugated version of OPG (OPG-Fc) or anti-RANKL, a cell-based bioassay employing a murine macrophage cell line (RAW 264.7), which expresses the receptor for RANK ligand, RANK, was developed. RAW 264.7 cells respond to RANK ligand by differentiating into osteoclast-like cells, expressing the terminal differentiation marker TRAP (tartrate-resistant acid phosphatase). Thus, the inhibition of RANK ligand by OPG-Fc or anti-RANKL would inhibit TRAP mRNA expression as well. However, if neutralizing antibodies to OPG-Fc or anti-RANKL antibodies were present, the TRAP mRNA would continue to be expressed.

Two assays were implements to determine whether there was a specific neutralizing activity to the protein therapeutic, a Screening NAb bioassay and Specificity bioassay. In the Screening NAb Bioassay patient serum was assessed for the presence of neutralizing antibodies to the protein therapeutic while the Specificity bioassay, the protein therapeutic and RANK ligand was used to eliminate false positives due to a non-specific induction of TRAP mRNA.

Example of Bioassay Format for Anti-RANK Ligand Antibody

For the Screening NAb bioassay, a sample containing 5% human patient serum, RANK Ligand and anti-RANKL antibody in cell Growth Media was used. Anti-RANK ligand antibody and RANK Ligand were sequentially added to the serum samples (NAb assay) with incubations for 30 minutes at 37⁰C following each addition RAW 264.7 cells (10,000 cells per well) were added to samples in Screening NAb and Specificity Assays. RAW 264.7 cells were added at 10,000 cells/well and this was incubated for 48 hours at 37° C. TRAP mRNA expression was detected using the Branched DNA Assay below.

For Specificity Bioassay, RAW 264.7 cells were added to a sample containing 5% patient serum only and incubated for 48 hours at 37° C. TRAP mRNA expression was detected using the Branched DNA Assay below.

The following samples were used for controls: Null Control (Control N), 5% pooled human serum; Maximum Control (Control M), 5% pooled human serum and RANK Ligand; Therapeutic Drug Control (Control D), 5% pooled human serum, RANK Ligand and anti-RANKL antibody or OPG; and Positive Control (Control P), 5% pooled human serum, RANK Ligand and anti-RANKL antibody and anti-anti-OPG ligand antibody (500 ng/mL in serum.

Branched DNA Assay. mRNA expression was measured using the Quantigene Screen kit commercially available from Genospectra, Inc. Briefly, RAW264.7 cells were lysed with a buffer containing Label Extender Probes, Blocking Probes and Capture Extender Probes. The cell lysate was transferred to a capture plate and was incubated overnight at 53⁰C. The plate was subsequently washed with a wash buffer (12.5 mL 20X SSC, 7.5 mL 0.01% Lithium Lauryl Sulfate, and 2.48 L water) followed by the addition of bDNA amplifier probe and incubated for 1 hour at 46⁰C. The plate was washed again with wash buffer and bDNA Label Probe was added, and incubated at 46⁰C for 1 hour. The plate was washed for final time with wash buffer followed by the addition of Substrate and a 30 minute incubation at 46⁰C. Luminescence was detected by TopCount NXT reader and measured in Counts per Second (CPS).

TABLE 6
bDNA Probes Used for Murine TRAP mRNA
Capture Extender Probes (CE)
aggagtgggagccatatgattTTTTTctcttggaaagaaagt
tggcgatctctttggcattggTTTTTctcttggaaagaaagt
agttccagcgcttggagatcTTTTTctcttggaaagaaagt
gtccgtgctcggcgatggaTTTTTctcttggaaagaaagt
tctcgtcctgaagatactgcaTTTTTctcttggaaagaaagt
tgaagccgcccagggagtcctcaTTTTTctcttggaaagaaagt
aagagtgattttccagaggcttTTTTTctcttggaaagaaagt
Label Extender Probes (LE)
acccatgaatccatccatcctggTTTTTaggcataggacccgtgtct
tgtaggcccagcagcagcaccTTTTTaggcataggacccgtgtct
gggctgtaccgtgggtcTTTTTaggcataggacccgtgtct
gggctgtaccgtgggtcTTTTTaggcataggacccgtgtct
ccccagtcgcccacagccaTTTTTaggcataggacccgtgtct
ccatttcccgggctgtgtggaTTTTTaggcataggacccgtgtct
tcgctggcatcgtgcactcTTTTTaggcataggacccgtgtct
cggtcagagaacacgtcctcTTTTTaggcataggacccgtgtct
tggtttccagccagcacataccTTTTTaggcataggacccgtgtct
agatggccacagttatgtttgTTTTTaggcataggacccgtgtct
gcatcactgtgtccagcataaTTTTTaggcataggacccgtgtct
aagtcatctgagttgccacacaTTTTTaggcataggacccgtgtct
ctgctgccaactgctttttgaTTTTTaggcataggacccgtgtct
cttgacaaggcagcgcgtggTTTTTaggcataggacccgtgtct
tggctaacaatggtcgcaagttTTTTTaggcataggacccgtgtct
ggttgtggtcatgtccacacaTTTTTaggcataggacccgtgtct
cagcacatagcccacaccgtTTTTTaggcataggacccgtgtct
tgatgtcgcacagagggatccTTTTTaggcataggacccgtgtct
Blocking Probes (BL)
caaatctcagggtgggagtgg
atggggcattggggacccct
cagagacatgatgaagtcagc
cagtgaagtagaaattgtcccc
aaaggtctcctggaacctcttg
aggggatgttgcgaagggca
tacgtggaattttgaagcgca
cattttgggctgctgctgactggca
gccaggacagctgagtgcgg
accaaaacgtagtcctccttgg
ccagatggggtagtggccggcc
ggtaggcagtgaccccgtatg
atgaagttgccggccccact
agccgttggggacctttcgt
gatccatagtgaaaccgcaagt
tggggcttatctccacatgtg

Results were analyzed using pre-determined criteria. Three ratios were used to determine the presence of neutralizing activity. The “NAb ratio” consisting of the mean sample CPS/mean CPS of the Therapeutic Drug Control (Control D) was used to screen for a the presence of any neutralizing activity to anti-RANK ligand antibody. The “Post/Pre” ratio consisting of the mean sample CPS of the post-dose sample/mean sample CPS of the pre-dose was used to determine the development of neutralizing activity between the pre and post-doses. The “Specificity Ratio”, consisting of the mean sample CPS of the Nab assay/mean sample CPS of the Specificity Assay, was used to determine if there were a factors in the serum inducing TRAP mRNA expression. In order for a sample to be considered positive for the presence of neutralizing activity as the result of a neutralizing antibody, a serum sample would be required to be found “Positive” in both the “Nab Ratio” and “Post/Pre Ratio,” and be found to not have non-anti-anti-RANK ligand antibody-specific TRAP gene expression.

Serum samples from 29 healthy donor volunteers were used to determine donor to donor variability and to derive assay thresholds for both the NAb and Specificity bioassays for the determination of a “positive” and “negative” sample. Results of the cell-based NAb assay are represented in FIG. 15. NAb assay threshold was determined as a ratio of Sample Mean CPS to Control D Mean CPS. Donors were spiked at 500 ng/mL of anti-anti-OPG ligand antibody in neat serum. All spiked donors were found to be above Nab Assay Threshold, and two unspiked donors were found to be above Nab Assay Threshold. FIG. 16 represents Validation of the Specificity Value Threshold. Specificity Value was determined as a ratio of Mean Raw CPS values generated in Nab Assay to Mean Raw CPS Values generated in the Specificity Assay. All spiked donors were found to be above the Specificity Value Threshold, whereas all unspiked donors were found to be below the Specificity Value Threshold, thus, no false negatives or false positives were generated.

TABLE 7
Alignment of mammalian EPO-R amino acid sequences
	SEQ
	ID	Alignment (numbering based on consensus sequence):
		1                                                   50
Mouse EPOR	NO:3	MdklrvplWP rVgpLCLLLA GAaWApsPsl PDpKFESKAA LLAsRGsEEL
Rat EPOR	NO:4	MdqlrvarWP rVspLCLLLA GAaWAssPsl PDpKFESKAA LLAsRGsEEL
Human EPOR	NO:2	MdhlgaslWP qVgsLCLLLA GAaWAppPnl PDpKFESKAA LLAaRGpEEL
Pig EPOR	NO:5	MyhfgatlWP gVgSLCLLLA GAtWApsPns PDaKFESKAA LLAaRGpEEL
Consensus	NO:1	M-------WP -V--LCLLLA GA-WA--P-- PD-KFESKAA LLA-RG-EEL
		51                                                 100
Mouse EPOR	NO:3	LCFTqRLEDL VCFWEEAass Gmd.fnYSFS YQLEgEsrKs CsLHQaPTvR
Rat EPOR	NO:4	LCFTqRLEDL VCFWEEAans Gmg.fnYSFS YQLEgEsrKs CrLHQaPTvR
Human EPOR	NO:2	LCFTeRLEDL VCFWEEAasa GVgpgnYSFS YQLEdEpWKl CrLHQaPTaR
Pig EPOR	NO:5	LCFTeRLEDL VCFWEEAgsa GvgpedYSFS YQLEgEpwKp ChLHQgPTaR
Consensus	NO:1	LCFT-RLEDL VCFWEEA--- G-----YSFS YQLE-E--K- C-LHQ-PT-R
		101                                                150
Mouse EPOR	NO:3	GsvRFWCSLP TADTSSFVPL ELqVTe.aSG sPRYHRiIHI NEVVLLDaPa
Rat EPOR	NO:4	GsmRFWCSLP TADTSSFVPL ELqVTe.aSG sPRYHRiIHI NEVVLLDaPa
Human EPOR	NO:2	GavRFWCSLP TADTSSFVPL ELrVT.aaSG aPRYHRvIHI NEVVLLDaPv
Pig EPOR	NO:5	GsvRFWCSLP TADTSSFVPL ELrVTevsSG aPRYHRiIHI NEVVLLDpPa
Consensus	NO:1	G--RFWCSLP TADTSSFVPL EL-VT---SG -PRYHR-IHI NEVVLLD-P-
		151                                                200
Mouse EPOR	NO:3	GLlARrAeEg sHVVLRWLPP PgaPMtthIR YEVdvSagNr AGgtQRVEvL
Rat EPOR	NO:4	GLlARrAeEg sHVVLRWLPP PgaPMtthIR YEVdvSagNr AGgtQRVEvL
Human EPOR	NO:2	GLvARlAdEs gHVVLRWLPP PetPMtshIR YEVdvSagNg AGsvQRVEiL
Pig EPOR	NO:5	GLlARrAeEs gHVVLRWLPP PgaPMaslIR YEVniSteNa AGgvQRVEiL
Consensus	NO:1	GL-AR-A-E- -HVVLRWLPP P--PM---IR YEV--S--N- AG--QRVE-L
		201                                                250
Mouse EPOR	NO:3	eGRTECVLSN LRGgTRYTFa VRARMAEPSF sGFWSAWSEP aSLLTaSDLD
Rat EPOR	NO:4	eGRTECVLSN LRGgTRYTFa VRARMAEPSF sGFWSAWSEP aSLLTaSDLD
Human EPOR	NO:2	eGRTECVLSN LRGrTRYTFa VRARMAEPSF gGFWSAWSEP vSLLTpSDLD
Pig EPOR	NO:5	dGRTECVLSN LRGgTRYTFm VRARMAEPSF gGFWSAWSEP aSLLTaSDLD
Consensus	NO:1	-GRTECVLSN LRG-TRYTF- VRARMAEPSF -GFWSAWSEP -SLLT-SDLD
		251                                                300
Mouse EPOR	NO:3	PLILTLSLIL VlIslLLtVL ALLSHRRtLq QKIWPGIPSP EsEFEGLFTT
Rat EPOR	NO:4	PLILTLSLIL VlIslLLtVL ALLSHRRaLr QKIWPGIPSP EnEFEGLFTT
Human EPOR	NO:2	PLILTLSLIL VvIlvLLtVL ALLSHRRaLk QKIWPGIPSP EsEFEGLFTT
Pig EPOR	NO:5	PLILTLSLIL VlIllLLaVL ALLSHRRtLk QKIWPGIPSP EgEFEGLFTT
Consensus	NO:1	PLILTLSLIL V-I--LL-VL ALLSHRR-L- QKIWPGIPSP E-EFEGLFTT
		301                                                350
Mouse EPOR	NO:3	HKGNFQLWLl QrDGCLWWSP gssFpEDPPA hLEVLSEprW avtQAgdpga
Rat EPOR	NO:4	HKGNFQLWLl QrDGCLWWSP sspFpEDPPA hLEVLSErrW gvtQAgdaga
Human EPOR	NO:2	HKGNFQLWLy QnDGCLWWSP ctpFtEDPPA sLEVLSErcW gtmQAvepgt
Pig EPOR	NO:5	HKGNFQLWLy QtDGCLWWSP ctpFaEDPPA pLEVLSErcW gvtQAvepaa
Consensus	NO:1	HKGNFQLWL- Q-DGCLWWSP ---F-EDPPA -LEVLSE--W ---QA-----
		351                                                400
Mouse EPOR	NO:3	dDeGpLLEPV GSEhAqDTYL VLDkWLLPRt PcSEnLsgPG gsvDpvtMDE
Rat EPOR	NO:4	eDkGpLLEPV GSErAqDTYL VLDeWLLPRc PcSEnLsgPG dsvDpatMDE
Human EPOR	NO:2	dDeGpLLEPV GSEhAqDTYL VLDkWLLPRn PpSEdLpgPG gsvDivaMDE
Pig EPOR	NO:5	dDeGsLLEPV GSEhArDTYL VLDkWLLPRr PaSEdLpqPG gdlDmaaMDE
Consensus	NO:1	-D-G-LLEPV GSE-A-DTYL VLD-WLLPR- P-SE-L--PG ---D---MDE
		401                                                450
Mouse EPOR	NO:3	aSEtSsCpSd LAsKPrPEGt SPsSFEYTIL DPSSqLLcPr aLppELPPTP
Rat EPOR	NO:4	gSEtSsCpSd LAsKPrPEGt SpsSFEYTIL DPSSkLLcPr aLppELPPTP
Human EPOR	NO:2	gSEaSsCsSa LAsKPsPEGa SaaSFEYTIL DPSSqLLrPw tLcpELPPTP
Pig EPOR	NO:5	aSEaSfCsSa LAlKPgPEGa SaaSFEYTIL DPSSqLLrPr aLpaELPPTP
Consensus	NO:1	-SE-S-C-S- LA-KP-PEG- S--SFEYTIL DPSS-LL-P- -L--ELPPTP
		451                                                500
Mouse EPOR	NO:3	PHLKYLYLVV SDSGISTDYS SGgSQgvhGd sSdGPYShPY ENSLvPdsEP
Rat EPOR	NO:4	PHLKYLYLVV SDSGISTDYS SCgSQgvhGd sSdGPYShPY ENSLvPdtEP
Human EPOR	NO:2	PHLKYLYLVV SDSGISTDYS SGdSQgaqGg lSdGPYSnPY ENSLiPaaEP
Pig EPOR	NO:5	PHLKYLYLVV SDSGISTDYS SGgSQetqGg sSsGPYSnPY ENSLvPapEP
Consensus	NO:1	PHLKYLYLVV SDSGISTDYS SG-SQ---G- -S-GPYS-PY ENSL-P--EP
		501   509
Mouse EPOR	NO:3	lhPgYVaCS
Rat EPOR	NO:4	lrPsYVaCS
Human EPOR	NO:2	lpPsYVaCS
Pig EPOR	NO:5	spPnYVtCS
Consensus	NO:1	--P-YV-CS

TABLE 8
Location of domains within EPO-R amino acid sequences; numbering refers
to amino acid positions within the corresponding SEQ ID NOs
	SEQ	Signal					“Mature”A
Polypeptide	ID	sequence	Extracellular	WSXWS	Transmembrane	Intracellular	polypeptide
Mouse EPOR	NO: 3	1-24	25-249	232-236	250-272	273-507	25-507
Rat EPOR	NO: 4	1-24	25-249	232-236	250-272	273-507	25-507
Human EPOR	NO: 2	1-24	25-250	233-237	251-273	274-508	25-508
Pig EPOR	NO: 5	1-24	25-251	234-238	252-274	275-509	25-509
Consensus	NO: 1	1-24	25-251	234-238	252-274	275-509	25-509
AFor the purposes of the above table, the “mature” polypeptide refers to a polypeptide from which the indicated signal sequence has been cleaved; additional mature polypeptide forms may occur.

TABLE 9
Alignment of mammalian EPO amino acid sequences
	SEQ
	ID	Alignment (numbering based on consensus sequence):
		1                                                   50
Mouse EPO	NO:10	MGvpe.rptl lLlLSllliP LGlPVlcAPp RLiCDSRVLE RYiLeAkEAE
Rat EPO	NO:1	MGvpe.rptl lLlLSllliP LGlPVlcAPp RLiCDSRVLE RYiLeAkEAE
Macaque EPO	NO:9	MGvhecpawl wLlLSlvslP LGlPVpgAPp RLiCDSRVLE RYlLeAkEAE
Rhesus EPO	NO:8	MGvheCpawl wLlLSlVslP LGlPVPgAPp RLvCDSRVLE RYlLeAkEAE
Human EPO	NO:7	MGvhecpawl wLlLSllslP LGlPVlgAPp RLiCDSRVLE RYlLeAkEAE
Cow EPO	NO:12	MGardctp.. lLmLSfllfp LGfPVlgAPa RLiCDSRVLE RYiLeArEAE
Sheep EPO	NO:13	MGardctpll lLlLSfllfP LGlpVlgAPp RLiCDSRVLE RYiLeArEAE
Cat EPO	NO:14	MGscec.pal lLlLSllllP LGlPVlgAPp RLiCDSRVLE RYiLgArEAE
Consensus	NO:6	MG-------- -L-LS----P LG-PV--AP- RL-CDSRVLE RY-L-A-EAE
		51                                                 100
Mouse EPO	NO:10	NvTmGCaEgp rlsENITVPD TKVNFYaWKR meVeeQAiEV WQGLsLLSEA
Rat EPO	NO:11	NvTmGCaEgp rlsENITVPD TKVNFYaWKR mkVeeQAvEV WQGLsLLSEA
Macaque EPO	NO:9	NvTmGCsEsc slnENITVPD TKVNFYaWKR meVgqQAvEV WQGLaLLSEA
Rhesus EPO	NO:8	NvTmGCsEsc slnENITVPD TKVNFYaWKR ieVgqQAvEV WQGLaLLSEA
Human EPO	NO:7	NiTtGCaEhc slnENITVPD TKVNFYaWKR meVgqQAvEV WQGLaLLSEA
Cow EPO	NO:12	NaTmGCaEgc sfnENITVPD TKVNFYaWKR meVqqQAlEV WQGLaLLSEA
Sheep EPO	NO:13	NaTmGCaEgc sfsENITVPD TKVNFYaWKR meVqqQAlEV WQGLaLLSEA
Cat EPO	NO:14	NvTmGCaEgc sfsENITVPD TKVNFYtWKR mdVgqQAvEV WQGLaLLSEA
Consensus	NO:6	N-T-GC-E-- ---ENITVPD TKVNFY-WKR --V--QA-EV WQGL-LLSEA
		101                                                150
Mouse EPO	NO:10	ilqaQAllaN sSQPpEtLqL HiDKAiSgLR SlTsLLRvLG AQkElmspPd
Rat EPO	NO:11	ilqaQAlqaN sSQPpEsLqL HiDKAiSgLR SlTsLLRvLG AQkElmspPd
Macaque EPO	NO:9	vlrgQAvlaN sSQPfEpLqL HmDKAiSgLR SiTtLLRaLG AQ.EaislPd
Rhesus EPO	NO:8	vlrgQAvlaN sSQPfEpLqL HmDKAiSgLR SiTtLLRaLG AQ.EaislPd
Human EPO	NO:7	vlrgQAllvN sSQPwEpLqL HvDKAvSgLR SlTtLLRaLG AQkEaispPd
Cow EPO	NO:12	ilrgQAllaN aSQPcEaLrL HvDKAvSgLR SlTsLLRaLG AQkEaislPd
Sheep EPO	NO:13	ifrgQAllaN aSQPcEaLrL HvDKAvSgLR SlTsLLRaLG AQkEaiplPd
Cat EPO	NO:14	ilrgQAllaN sSQPsEtLqL HvDKAvSsLR SlTsLLRaLG AQkEatslPe
Consensus	NO:6	----QA---N -SQP-E-L-L H-DKA-S-LR S-T-LLR-LG AQ-E----P-
		151                                         194
Mouse EPO	NO:10	ttp.pAPLRt lTvDtfcKLF RvYaNFLRGK LkLYTGEvCR rGDR
Rat EPO	NO:11	atq.aAPLRt lTaDtfcKLF RvYsNFLRGK LkLYTGEaCR rGDR
Macague EPO	NO:9	aa.saAPLRt iTaDtfcKLF RvYsNFLRGK LkLYTGEaCR rGDR
Rhesus EPO	NO:8	aa.saAPLRt iTaDtfcKLF RvYSNFLRGK LkLYTGEaCR rGDR
Human EPO	NO:7	aa.saAPLRt iTaDtfrKLF RvYsNFLRGK LkLYTGEaCR tGDR
Cow EPO	NO:12	atpsaAPLRa fTvDalsKLF RiYsNFLRGK LtLYTGEaCR rGDR
Sheep EPO	NO:13	atpsaAPLRi fTvDalsKLF RiYsNFLRGK LtLYTGEaCR rGDR
Cat EPO	NO:14	at.saAPLRt fTvDtlcKLF RiYsNFLRGK LtLYTGEaCR rGDR
Consensus	NO:6	-----APLR- -T-D---KLF R-Y-NFLRGK L-LYTGE-CR -GDR

TABLE 10
Location of domains within EPO amino acid sequences; numbering
refers to amino acid positions within the corresponding SEQ ID NOs
Polypeptide	SEQ ID	Signal sequence	“Mature”A polypeptide
Mouse EPO	NO: 10	1-26	27-192
Rat EPO	NO: 11	1-26	27-192
Macaque EPO	NO: 9	1-27	28-192
Rhesus EPO	NO: 8	1-27	28-192
Human EPO	NO: 7	1-27	28-193
Cow EPO	NO: 12	1-25	26-192
Sheep EPO	NO: 13	1-27	28-194
Cat EPO	NO: 14	1-26	27-192
Consensus	NO: 6	1-27	28-194
AFor the purposes of the above table, the “mature” polypeptide refers to a polypeptide from which the indicated signal sequence has been cleaved; additional mature polypeptide forms may occur.

TABLE 11
Alignment of mammalian PIM1 coding (cDNA) nucleotide
sequences
	SEQ
	ID	Alignment (numbering based on consensus sequence):
		1                                                    50
Mouse PIM1	NO:21	ATGCTCcTGT CCAAgATCAA CTCcCTgGCC CACCTGCGCg CcGCgCCcTG
Rat PIM1	NO:20	ATGCTCtTGT CCAAgATCAA CTCcCTgGCC CACCTGCGCg CaGCcCCtTG
Cat PIM1	NO:18	ATGCTCtTGT CCAAaATCAA CTCgCTtGCC CACCTGCGCa CcGCgCCcTG
Human PIM1	NO:16	ATGCTCtTGT CCAAaATCAA CTCgCTtGCC CACCTGCGCg CcGCgCCcTG
Cow PIM1	NO:19	ATGCTCtTGT CCAAaATCAA CTCgCTtGCC CACCTGCGCg CcGCgCCcTG
Chimp PIM1	NO:17	ATGCTCtTGT CCAAaATCAA CTCgCTtGCC CACCTGCGCg CcGCgCCcTG
ConsensusA	NO:15	ATGCTCYTGT CCAARATCAA CTCSCTKGCC CACCTGCGCR CMGCSCCYTG
		51                                                 100
Mouse PIM1	NO:21	CAaCGACCTG CACGCCAcCA AGCTGGCGCC gGGCAAaGAG AAGGAGCCCC
Rat PIM1	NO:20	CAaCGACCTG CACGCCAaCA AGCTGGCGCC gGGCAAaGAG AAGGAGCCCC
Cat PIM1	NO:18	CAaCGACCTG CACGCCAcCA AGCTGGCGCC cGGCAAgGAG AAGGAGCCCC
Human PIM1	NO:16	CAaCGACCTG CACGCCAcCA AGCTGGCGCC cGGCAAgGAG AAGGAGCCCC
Cow PIM1	NO:19	CAgCGACCTG CACGCCAcCA AGCTGGCGCC gGGCAAgGAG AAGGAGCCCC
Chimp PIM1	NO:17	CAaCGACCTG CACGCCAcCA AGCTGGCGCC cGGCAAgGAG AAGGAGCCCC
Consensus	NO:15	CARCGACCTG CACGCCAMCA AGCTGGCGCC SGGCAARGAG AAGGAGCCCC
		101                                                150
Mouse PIM1	NO:21	TGGAGTCGCA GTACCAGGTG GGCCCGCTgt TGGGCAGcGG tGGCTTCGGC
Rat PIM1	NO:20	TGGAGTCGCA GTACCAGGTG GGCCCGCTgt TGGGCAGcGG tGGCTTCGGC
Cat PIM1	NO:18	TGGAGTCGCA GTACCAGGTG GGCCCGCTac TGGGCAGcGG cGGCTTCGGC
Human PIM1	NO:16	TGGAGTCGCA GTACCAGGTG GGCCCGCTac TGGGCAGcGG cGGCTTCGGC
Cow PIM1	NO:19	TGGAGTCGCA GTACCAGGTG GGCCCGCTcc TGGGCAGtGG cGGCTTCGGC
Chimp PIM1	NO:17	TGGAGTCGCA GTACCAGGTG GGCCCGCTac TGGGCAGcGG cGGCTTCGGC
Consensus	NO:15	TGGAGTCGCA GTACCAGGTG GGCCCGCTVY TGGGCAGYGG YGGCTTCGGC
		151                                                200
Mouse PIM1	NO:21	TCGGTcTACT CtGGCATCCG cGTCgCCGAC AACTTGCCGG TGGCCATtAA
Rat PIM1	NO:20	TCGGTcTACT CgGGCATCCG cGTCgCCGAC AACTTGCCGG TGGCCATcAA
Cat PIM1	NO:18	TCGGTcTACT CaGGCATCCG gGTCgCCGAC AACTTGCCGG TGGCCATcAA
Human PIM1	NO:16	TCGGTcTACT CaGGCATCCG cGTCtCCGAC AACTTGCCGG TGGCCATcAA
Cow PIM1	NO:19	TCGGTgTACT CaGGCATCCG tGTCgCCGAC AACTTGCCGG TGGCCATcAA
Chimp PIM1	NO:17	TCGGTcTACT CaGGCATCCG cGTCtCCGAC AACTTGCCGG TGGCCATcAA
Consensus	NO:15	TCGGTSTACT CDGGCATCCG BGTCKCCGAC AACTTGCCGG TGGCCATYAA
		201                                                250
Mouse PIM1	NO:21	gCACGTGGAG AAGGACCGGA TTTCCGAtTG GGGaGAaCTG CCcAAtGGCA
Rat PIM1	NO:20	gCACGTGGAG AAGGACCGGA TTTCCGAcTG GGGgGAaCTG CCcAAcGGCA
Cat PIM1	NO:18	gCACGTGGAG AAGGACCGGA TTTCCGAtTG GGGaGAgCTG CCcAAtGGCA
Human PIM1	NO:16	aCACGTGGAG AAGGACCGGA TTTCCGAcTG GGGaGAgCTG CCtAAtGGCA
Cow PIM1	NO:19	gCACGTGGAG AAGGACCGGA TTTCCGAcTG GGGaGAgCTG CCtAAtGGCA
Chimp PIM1	NO:17	aCACGTGGAG AAGGACCGGA TTTCCGAcTG GGGaGAgCTG CCtAAtGGCA
Consensus	NO:15	RCACGTGGAG AAGGACCGGA TTTCCGAYTG GGGRGARCTG CCYAAYGGCA
		251                                                300
Mouse PIM1	NO:21	CcCGAGTGCC CATGGAaGTG GTcCTGtTGA AGAAGGTGAG CTCGGacTTC
Rat PIM1	NO:20	CcCGAGTGCC CATGGAaGTG GTcCTGcTGA AGAAGGTGAG CTCGGgcTTC
Cat PIM1	NO:18	CcCGAGTGCC CATGGAgGTG GTcCTGcTGA AGAAGGTGAG CTCGGgcTTC
Human PIM1	NO:16	CtCGAGTGCC CATGGAaGTG GTcCTGcTGA AGAAGGTGAG CTCGGgtTTC
Cow PIM1	NO:19	CcCGAGTGCC CATGGAaGTG GTtCTGcTGA AGAAGGTGAG CTCGGgcTTC
Chimp PIM1	NO:17	CtCGAGTGCC CATGGAaGTG GTcCTGcTGA AGAAGGTGAG CTCGGgtTTC
Consensus	NO:15	CYCGAGTGCC CATGGARGTG CTYCTGYTGA AGAAGGTGAG CTCGGRYTTC
		301                                                350
Mouse PIM1	NO:21	TCgGGCGTCA TTaGaCTtCT GGACTGGTTc GAGAGGCCCG AtAGTTTCGT
Rat PIM1	NO:20	TCgGGCGTCA TTaGaCTtCT GGACTGGTTc GAGAGGCCCG AtAGTTTCGT
Cat PIM1	NO:18	TCcGGCGTCA TTcGgCTcCT GGACTGGTTt GAGAGGCCCG AcAGTTTCGT
Human PIM1	NO:16	TCcGGCGTCA TTaGgCTcCT GGACTGGTTc GAGAGGCCCG AcAGTTTCGT
Cow PIM1	NO:19	TCcGGCGTCA TTaGgCTcCT GGACTGGTTc GAGAGGCCCG AcAGTTTCGT
Chimp PIM1	NO:17	TCcGGCGTCA TTaGgCTcCT GGACTGGTTc GAGAGGCCCG AcAGTTTCGT
Consensus	NO:15	TCSGGCGTCA TTMGRCTYCT GGACTGGTTY GAGAGGCCCG AYAGTTTCGT
		351                                                400
Mouse PIM1	NO:21	gcTGATCCTG GAGAGGCCcG AaCCgGTGCA AGAcCTCTTC GACTTtATCA
Rat PIM1	NO:20	gcTGATCCTG GAGAGGCCcG AaCCcGTGCA AGAcCTCTTC GACTTcATCA
Cat PIM1	NO:18	ctTGATCCTG GAGAGGCCcG AgCCgGTGCA AGAcCTCTTC GACTTtATCA
Human PIM1	NO:16	ccTGATCCTG GAGAGGCCcG AgCCgGTGCA AGAtCTCTTC GACTTcATCA
Cow PIM1	NO:19	ccTGATCCTG GAGAGGCCgG AgCCgGTGCA AGAcCTCTTC GACTTtATCA
Chimp PIM1	NO:17	ccTGATCCTG GAGAGGCCcG AgCCgGTGCA AGAtCTCTTC GACTTtATCA
Consensus	NO:15	SYTGATCCTG GAGAGGCCSG ARCCSGTGCA AGAYCTCTTC GACTTYATCA
		401                                                450
Mouse PIM1	NO:21	CcGAacGaGG aGCcCTaCAg GAGGAcCTgG CCCGagGaTT CTTCTGGCAG
Rat PIM1	NO:20	CcGAgcGaGG aGCcCTcCAg GAGGAgCTgG CCCGgaGcTT CTTCTGGCAG
Cat PIM1	NO:18	CgGAaaGgGG gGCtCTgCAg GAGGAgCTgG CCCGcaGcTT CTTCTGGCAG
Human PIM1	NO:16	CgGAaaGgGG aGCcCTgCAa GAGGAgCTcG CCCGcaGcTT CTTCTGGCAG
Cow PIM1	NO:19	CgGAaaGgGG gGCtCTgCAg GAGGAgCTgG CCCGcaGcTT CTTCTGGCAG
Chimp PIM1	NO:17	CgGAaaGgGG gGCcCTgCAa GAGGAgCTgG CCCGcaGcTT CTTCTGGCAG
Consensus	NO:15	CSGARMGRCG RGCYCTVCAR GAGGASCTSG CCCGVRGMTT CTTCTGGCAG
		451                                                500
Mouse PIM1	NO:21	GTgCTGGAGG CcGTGCGgCA tTGCCACaAC TGCGGGGTtC TcCACCGCGA
Rat PIM1	NO:20	GTgCTGGAGG CcGTGCGgCA tTGCCACaAC TGCGGGGTtC TcCACCGCGA
Cat PIM1	NO:18	GTgCTGGAGG CcGTGCGgCA cTGCCACaAC TGCGGGGTgC TcCACCGCGA
Human PIM1	NO:16	GTgCTGGAGG CcGTGCGgCA cTGCCACaAC TGCGGGGTgC TcCACCGCGA
Cow PIM1	NO:19	GTaCTGGAGG CgGTGCGaCA cTGCCACgAC TGCGGGGTgC TtCACCGCGA
Chimp PIM1	NO:17	GTgCTGGAGG CcGTGCGgCA cTGCCACaAC TGCGGGGTgC TcCACCGCGA
Consensus	NO:15	GTRCTGGAGG CSGTGCGRCA YTGCCACRAC TGCGGGGTKC TYCACCGCGA
		501                                                550
Mouse PIM1	NO:21	CATCAAGGAC GAgAACATCt TaATCGACCT gAgcCGCGGC GAaaTCAAaC
Rat PIM1	NO:20	CATCAAGGAC GAgAACATCt TaATCGACCT gAacCGCGGC GAacTCAAaC
Cat PIM1	NO:18	CATCAAGGAC GAgAACATCc TcATCGACCT cAatCGCGGC GAgcTCAAgC
Human PIM1	NO:16	CATCAAGGAC GAaAACATCc TtATCGACCT cAatCGCGGC GAgcTCAAgC
Cow PIM1	NO:19	CATCAAGGAC GAgAACATCc TtATCGACCT cAatCGCGGC GAgcTCAAgC
Chimp PIM1	NO:17	CATCAAGGAC GAaAACATCc TtATCGACCT cAatCGCGGC GAgcTCAAgC
Consensus	NO:15	CATCAAGGAC GARAACATCY THATCGACCT SARYCGCGGC GARMTCAARC
		551                                                600
Mouse PIM1	NO:21	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AgGACACaGT CTACACGGAC
Rat PIM1	NO:20	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AgGACACaGT CTACACGGAC
Cat PIM1	NO:18	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AgGACACcGT CTACACGGAC
Human PIM1	NO:16	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AaGACACcGT CTACACGGAC
Cow PIM1	NO:19	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AgGACACcGT CTACACGGAC
Chimp PIM1	NO:17	TCATCGACTT CGGGTCGGGG GCGCTGCTCA AgGACACcGT CTACACGGAC
Consensus	NO:15	TCATCGACTT CGGGTCGGGG GCGCTGCTCA ARGACACMGT CTACACGGAC
		601                                                650
Mouse PIM1	NO:21	TTtGAtGGgA CCCGAGTGTA cAGtCCtCCA GAGTGGATtC GCTAcCATCG
Rat PIM1	NO:20	TTtGAcGGaA CCCGAGTGTA cAGtCCtCCA GAGTGGATtC GCTAcCATCG
Cat PIM1	NO:18	TTcGAcGGgA CCCGAGTGTA tAGtCCcCCA GAGTGGATcC GCTAcCATCG
Human PIM1	NO:16	TTcGAtGGgA CCCGAGTGTA tAGcCCtCCA GAGTGGATcC GCTAcCATCG
Cow PIM1	NO:19	TTcGAtGGgA CCCGAGTGTA tAGtCCtCCA GAGTGGATcC GCTAtCATCG
Chimp PIM1	NO:17	TTcGAtGGgA CCCGAGTGTA tAGcCCtCCA GAGTGGATcC GCTAcCATCG
Consensus	NO:15	TTYGAYGGRA CCCGAGTGTA YAGYCCYCCA GAGTGGATYC GCTAYCATCG
		651                                                700
Mouse PIM1	NO:21	CTACCAcGGC AGGTCGGCaG CtGTcTGGTC cCTtGGGATC CTGCTcTATG
Rat PIM1	NO:20	CTACCAcGGC AGGTCGGCtG CtGTtTGGTC cCTgGGGATC CTGCTcTATG
Cat PIM1	NO:18	CTACCAtGGC AGGTCGGCcG CcGTcTGGTC tCTgGGGATC CTGCTgTATG
Human PIM1	NO:16	CTACCAtGGC AGGTCGGCgG CaGTcTGGTC cCTgGGGATC CTGCTgTATG
Cow PIM1	NO:19	CTACCAtGGC AGGTCGGCaG CcGTcTGGTC tCTgGGGATC CTGCTgTATG
Chimp PIM1	NO:17	CTACCAtGGC AGGTCGGCgG CaGTcTGGTC cCTgGGGATC CTGCTgTATG
Consensus	NO:15	CTACCAYGGC AGGTCGGCNG CHGTYTGGTC YCTKGGGATC CTGCTSTATG
		701                                                750
Mouse PIM1	NO:21	AcATGgTctg cGGaGAtaTt ccgtttgagc acgatgaaga gatcaTCaag
Rat PIM1	NO:20	AcATGgTctg cGGaGAtaTt ccatttgagc acgacgaaga gatcgTCaag
Cat PIM1	NO:18	AtATGgTctg tGGaGAtaTt ccttttgagc atgatgaaga gatcaTCagg
Human PIM1	NO:16	AtATGgTgtg tGGaGAtaTt cctttcgagc atgacgaaga gatcaTCagg
Cow PIM1	NO:19	AcATGgTgtg cGGaGAtaTt ccctttgagc acgatgagga gattgTCagg
Chimp PIM1	NO:17	AtATGtTggt aGGtGAatTg aatcatctca tcatgctcag tggtgTCtca
Consensus	NO:15	AYATGKTSKK HGGWGAWWTK MMNYWYSWSM WYRWBSWVRR KRKYRTCWVR
		751                                                800
Mouse PIM1	NO:21	ggccAagtgt tctTcAggca aactgtctct TCaGAgTGTC AgCAcCTtAT
Rat PIM1	NO:20	ggccAagtgt actTtAggca aagggtctct TCaGAaTGTC AaCAtCTtAT
Cat PIM1	NO:18	ggccAagttt tctTcAggca gagggtctct TCaGAgTGTC AgCAtCTcAT
Human PIM1	NO:16	ggccAggttt tctTcAggca gagggtctct TCaGAaTGTC AgCAtCTcAT
Cow PIM1	NO:19	ggccAagttt tctTcAggca gcgggtctcc TCaGAgTGTC AaCAtCTcAT
Chimp PIM1	NO:17	tcaaAatctc ttgTcAtcat ccttcctatt TCtGAaTGTC AgCAtCTcAT
Consensus	NO:15	KSMMARKYKY WYKTYAKSMW VMBKSYYWYY TCWGARTGTC ARCAYCTYAT
		801                                                850
Mouse PIM1	NO:21	TAaATGGTGC cTGtCCcTGA GACCaTCaGA tcGGCCctCC TTtGAAGAAA
Rat PIM1	NO:20	TAgATGGTGC cTGtCCcTGA GACCaTCgGA ccGGCCctCC TTtGAAGAAA
Cat PIM1	NO:18	TAgATGGTGC tTGgCCcTGA GACCgTCaGA ccGGCCatCC TTcGAAGAAA
Human PIM1	NO:16	TAgATGGTGC tTGgCCcTGA GACCaTCaGA taGGCCaaCC TTcGAAGAAA
Cow PIM1	NO:19	TAgATGGTGC tTGgCCtTGA GACCaTCaGA tcGGCCaaCC TTcGAAGAAA
Chimp PIM1	NO:17	TAgATGGTGC tTGgCCcTGA GACCaTCaGA taGGCCaaCC TTcGAAGAAA
Consensus	NO:15	TARATGGTGC YTGKCCYTGA GACCRTCRGA YMGGCCMWCC TTYGAAGAAA
		851                                                900
Mouse PIM1	NO:21	TCCgGAACCA TCCaTGGATG CAgGgtGacC TCCTGCCCCA GGcagCttCt
Rat PIM1	NO:20	TCCaGAACCA TCCgTGGATG CAgGatGttC TCCTGCCCCA GGccaCcgCc
Cat PIM1	NO:18	TCCaGAACCA TCCcTGGATG CAaGatGtcC TCCTGCCCCA GGaaaCagCc
Human PIM1	NO:16	TCCaGAACCA TCCaTGGATG CAaGatGttC TCCTGCCCCA GGaaaCtgCt
Cow PIM1	NO:19	TCCaGAACCA TCCgTGGATG CAaGacGtcC TCCTGCCCCA GGaaaCtgCt
Chimp PIM1	NO:17	TCCaGAACCA TCCaTGGATG CAaGatGttC TCCTGCCCCA GGaaaCtgCt
Consensus	NO:15	TCCRGAACCA TCCVTGGATG CARGRYGWYC TCCTGCCCCA GGMMRCHKCY
		901                                        942
Mouse PIM1	NO:21	GAGATcCAtC TgCACAGtCT GTCaCCgggg tCCAGCAAgT AG
Rat PIM1	NO:20	GAGATtCAtC TgCACAGcCT GTCaCCatca cCCAGCAAaT AG
Cat PIM1	NO:18	GAGATCCAtC TgCACAGcCT GTCaCCaggg cCCAGCAAaT AG
Human PIM1	NO:16	GAGATcCAcC TcCACAGcCT GTCgCCgggg cCCAGCAAaT AG
Cow PIM1	NO:19	GAGATcCAtC TcCACAGcCT GTCgCCaggg cCCAGCAAaT AG
Chimp PIM1	NO:17	GAGATcCAcC TcCACAGcCT GTCgCCgggg cCCAGCAAaT AG
Consensus	NO:15	GAGATYCAYC TSCACAGYCT GTCRCCRKSR YCCAGCAART AG
AThe symbols in the Consensus sequence conform to Annex C, Appendix 2, TABLE 1 of the Patent Cooperation Treaty Administrative Instructions.

TABLE 12
Alignment of mammalian NGF-R amino acid
sequences
	SEQ
	ID:	Alignment (numbering based on consensus sequence):
		1                                                   50
Mouse NGFR	NO:82	MLRGqRlGQL GWHrpAAGlG sLmtsLmLAc AsAAsCrevC CPvGpSGLRC
Rat NGFR	NO:83	MLRGqRhGQL GWHrpAAGlG gLvtsLmLAc AcAAsCretC CPvGpSGLRC
Human NGFR	NO:81	MLRGgRrGQL GWHswAAGpG sLlawLiLAs AgAApCpdaC CPhGsSGLRC
Consensus	NO:80	MLRG-R-GQL GWH--AAG-G -L---L-LA- A-AA-C---C CP-G-SGLRC
		51                                                 100
Mouse NGFR	NO:82	TRaGsLdtLr gLrGAgNLTE LYvENQqhLQ rLEfeDLqGL GELRsLTIVK
Rat NGFR	NO:83	TRaGtLntLr gLrGAgNLTE LYvENQrdLQ rLEfeDLqGL GELRsLTIVK
Human NGFR	NO:81	TRdGaLdsLh hLpGAeNLTE LYiENQqhLQ hLElrDLrGL GELRnLTIVK
Consensus	NO:80	TR-G-L--L- -L-GA-NLTE LY-ENQ--LQ -LE--DL-GL GELR-LTIVK
		101                                                150
Mouse NGFR	NO:82	SGLRFVAPDA FrFTPRLShL NLSsNALESL SWKTVQGLSL QdLtLSGNPL
Rat NGFR	NO:83	SGLRFVAPDA FhFTPRLShL NLSsNALESL SWKTVQGLSL QdLtLSGNPL
Human NGFR	NO:81	SGLRFVAPDA FhFTPRLSrL NLSfNALESL SWKTVQGLSL QeLvLSGNPL
Consensus	NO:80	SGLRFVAPDA F-FTPRLS-L NLS-NALESL SWKTVQGLSL Q-L-LSGNPL
		151                                                200
Mouse NGFR	NO:82	HCSCALfWLQ RWEqEgLcGV htQtLhdsGp GdqflPLgH. .NtSCGVPtv
Rat NGFR	NO:83	HCSCALlWLQ RWEqEdLcGV ytQkLqgsGs GdqflPLgH. .NnSCGVPsv
Human NGFR	NO:81	HCSCALrWLQ RWEeEgLgGV peQkLqchGq G....PLaHm pNaSCGVPtl
Consensus	NO:80	HCSCAL-WLQ RWE-E-L-GV --Q-L---G- G----PL-H- -N-SCGVP--
		201                                                250
Mouse NGFR	NO:82	KiQmPNdSVe VGDDVfLqCQ VEGlaLqQAd WILTELEgaA TvkKfGdLPS
Rat NGFR	NO:83	KiQmPNdSVe VGDDVfLgCQ VEGqaLqQAd WILTELEgtA TmkKsGdLPS
Human NGFR	NO:81	KvQvPNaSVd VGDDVlLrCQ VEGrgLeQAg WILTELEqsA TvmKsGgLPS
Consensus	NO:80	K-Q-PN-SV- VGDDV-L-CQ VEG--L-QA- WILTELE--A T--K-C-LPS
		251                                                300
Mouse NGFR	NO:82	LGLiLvNVTS DLNkKNvTCW AENDVGRAEV SVQVsVSFPA SVhLglAVEq
Rat NGFR	NO:83	LGLtLvNVTS DLNkKNvTCW AENDVGRAEV SVQVsVSFPA SVhLgkAVEq
Human NGFR	NO:81	LGLtLaNVTS DLNrKNlTCW AENDVGRAEV SVQVnVSFPA SVqLhtAVEm
Consensus	NO:80	LGL-L-NVTS DLN-KN-TCW AENDVGRAEV SVQV-VSFPA SV-L--AVE-
		301                                                350
Mouse NGFR	NO:82	HHWCIPFSVD GQPAPSLRWl FNGSVLNETS FIFTqFLEsA ltNETmRHGC
Rat NGFR	NO:83	HHWCIPFSVD GQPAPSLRWf FNGSVLNETS FIFTqFLEsA ltNETmRHGC
Human NGFR	NO:81	HHWCIPFSVD GQPAPSLRWl FNGSVLNETS FIFTeFLEpA .aNETvRHGC
Consensus	NO:80	HHWCIPFSVD GQPAPSLRW- FNGSVLNETS FIFT-FLE-A --NET-RHGC
		351                                                400
Mouse NGFR	NO:82	LRLNQPTHVN NGNYTLLAAN PyGQAaASvM AAFMDNPFEF NPEDPIPVSF
Rat NGFR	NO:83	LRLNQPTHVN NGNYTLLAAN PyGQAaASiM AAFMDNPFEF NPEDPIPVSF
Human NGFR	NO:81	LRLNQPTHVN NGNYTLLAAN PfGQAsASiM AAFMDNPFEF NPEDPIPVSF
Consensus	NO:80	LRLNQPTHVN NGNYTLLAAN P-GQA-AS-M AAFMDNPFEF NPEDPIPVSF
		401                                                450
Mouse NGFR	NO:82	SPVDgNSTSr DPVEKKDETP FGVSVAVGLA VsAaLFLSaL LLVLNKCGqR
Rat NGFR	NO:83	SPVDtNSTSr DPVEKKDETP FGVSVAVGLA VsAaLFLSaL LLVLNKCGqR
Human NGFR	NO:81	SPVDtNSTSg DPVEKKDETP FGVSVAVGLA VfAcLFLStL LLVLNKCGrR
Consensus	NO:80	SPVD-NSTS- DPVEKKDETP FGVSVAVGLA V-A-LFLS-L LLVLNKCG-R
		451                                                500
Mouse NGFR	NO:82	sKFGINRPAV LAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHImENPQ
Rat NGFR	NO:83	sKFGINRPAV LAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHImENPQ
Human NGFR	NO:81	nKFGINRPAV LAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHIiENPQ
Consensus	NO:80	-KFGINRPAV LAPEDGLAMS LHFMTLGGSS LSPTEGKGSG LQGHI-ENPQ
		501                                                550
Mouse NGFR	NO:82	YFSDtCVHHI KRqDIiLKWE LGEGAFGKVF LAECyNLLnd QDKMLVAVKA
Rat NGFR	NO:83	YFSDtCVHHI KRqDIiLKWE LGEGAFGKVF LAECyNLLnd QDKMLVAVKA
Human NGFR	NO:81	YFSDaCVHHI KRrDIvLKWE LGEGAFGKVF LAEChNLLpe QDKMLVAVKA
Consensus	NO:80	YFSD-CVHHI KR-DI-LKWE LGEGAFGKVF LAEC-NLL-- QDKMLVAVKA
		551                                                600
Mouse NGFR	NO:82	LKEaSEnARQ DFqREAELLT MLQHQHIVRF FGVCTEGgPL LMVFEYMRHG
Rat NGFR	NO:83	LKEtSEnARQ DFhREAELLT MLQHQHIVRF FGVCTEGgPL LMVFEYMRHG
Human NGFR	NO:81	LKEaSEsARQ DFqREAELLT MLQHQHIVRF FGVCTEGrPL LMVFEYMRHG
Consensus	NO:80	LKE-SE-ARQ DF-REAELLT MLQHQHIVRF FGVCTEG-PL LMVFEYMRHG
		601                                                650
Mouse NGFR	NO:82	DLNRFLRSHG PDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLAsLH
Rat NGFR	NO:83	DLNRFLRSHG PDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLAsLH
Human NGFR	NO:81	DLNRFLRSHG PDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLAgLH
Consensus	NO:80	DLNRFLRSHG PDAKLLAGGE DVAPGPLGLG QLLAVASQVA AGMVYLA-LH
		651                                                700
Mouse NGFR	NO:82	FVHRDLATRN CLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP
Rat NGFR	NO:83	FVHRDLATRN CLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP
Human NGFR	NO:81	FVHRDLATRN CLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP
Consensus	NO:80	FVHRDLATRN CLVGQGLVVK IGDFGMSRDI YSTDYYRVGG RTMLPIRWMP
		701                                                750
Mouse NGFR	NO:82	PESILYRKFs TESDVWSFGV VLWEIFTYGK QPWYQLSNTE AIeCITQGRE
Rat NGFR	NO:83	PESILYRKFs TESDVWSFGV VLWEIFTYGK QPWYQLSNTE AIeCITQGRE
Human NGFR	NO:81	PESILYRKFt TESDVWSFGV VLWEIFTYGK QPWYQLSNTE AIdCITQGRE
Consensus	NO:80	PESILYRKF- TESDVWSFGV VLWEIFTYGK QPWYQLSNTE AI-CITQGRE
		751                                                801
Mouse NGFR	NO:82	LERPRACPPd VYAIMRGCWQ REPQQRlSmK DVHARLQALA QAPPsYLDVLG
Rat NGFR	NO:83	LERPRACPPd VYAIMRGCWQ REPQQRlSmK DVHARLQALA QAPPsYLDVLG
Human NGFR	NO:81	LERPRACPPe VYAIMRGCWQ REPQQRhSiK DVHARLQALA QAPPvYLDVLG
Consensus	NO:80	LERPRACPP- VYAIMRGCWQ REPQQR-S-K DVHARLQALA QAPP-YLDVLG

TABLE 13
Location of domains within NGF-R amino acid sequences; numbering refers
to amino acid positions within the corresponding SEQ ID NOs
	SEQ	Signal				“Mature”A
Polypeptide	ID	sequence	Extracellular	Transmembrane	Intracellular	polypeptide
Mouse NGFR	NO: 82	1-32	33-418	419-442	443-799	33-799
Rat NGFR	NO: 83	1-32	33-418	419-442	443-799	33-799
Human NGFR	NO: 81	1-32	33-415	416-439B	440-796	33-796
Consensus	NO: 80	1-32	33-420	421-444	445-801	33-801
AFor the purposes of the above table, the “mature” polypeptide refers to a polypeptide from which the indicated signal sequence has been cleaved; additional mature polypeptide forms may occur.
BIn the table above, the location of the transmembrane domain in the human NGF-R amino acid sequence is shown as corresponding to the location of the transmembrane domain in the other mammalian NGF-R sequences.

TABLE 14
Alignment of mammalian NGF amino acid sequences
	SEQ
	ID	Alignment (numbering based on consensus sequence):
		1                                                   50
Gorilla NGF	NO:86	MSMLFYTLIT afLIGiQAEl hseSNvpaGh tiPqaHWTKL QHSLDTALRR
Orang. NGF	NO:87	MSMLFYTLIT afLIGiQAEp hseSNvpaGh tiPqaHWTKL QHSLDTALRR
Human NGF	NO:85	MSMLFYTLIT afLIGiQAEp hseSNvpaGh tiPqvHWTKL QHSLDTALRR
Mouse NGF	NO:89	MSMLFYTLIT afLIGvQAEp ytdSNvpeGd svPeaHWTKL QHSLDTALRR
Rat NGF	NO:88	MSMLFYTLIT afLIGvQAEp ytdSNvpeGd svPeaHWTKL QHSLDTALRR
Aft. rat NGF	NO:91	MSMLFYTLIT alLIGvQAEp ytdSNlpeGd svPeaHWTKL QHSLDTALRR
G. pig NGF	NO:90	MSMLFYTLIT vfLIGiQAEp ysdSNvlsGd tiPqaHWTKL QHSLDTALRR
Consensus	NO:84	MSMLFYTLIT --LIG-QAE- ---SN---G- --P--HWTKL QHSLDTALRR
		51                                                 100
Gorilla NGF	NO:86	ArSaPaaaIA ARVaGQTrNI TVDPrLFKKR rLrSPRVLFS TQPPpeaaDt
Orang. NGF	NO:87	ArStPaaaIA ARVaGQTcNI TVDPrLFKKR rLrSPRVLFS TQPPpeaaDt
Human NGF	NO:85	ArSaPaaaIA ARVaGQTrNI TVDPrLFKKR rLrSPRVLFS TQPPreaaDt
Mouse NGF	NO:89	ArSaPtapIA ARVtGQTrNI TVDPrLFKKR rLhSPRVLFS TQPPptssDt
Rat NGF	NO:88	ArSaPaepIA ARVtGQTrNI TVDPkLFKKR rLrSPRVLFS TQPPptssDt
Afr. rat NGF	NO:91	ArSaPaapIA ARVtGQTrNI TVDPrLFKKR kLrSPRVLFS TQPPptssDt
G. pig NGF	NO:90	AhSaPaapIA ARVaGQTlNI TVDPrLFKKR rLhSPRVLFS TQPPplstDa
Consensus	NO:84	A-S-P---IA ARV-GQT-NI TVDP-LFKKR -L-SPRVLFS TQPP- - -
		101                                                150
Gorilla NGF	NO:86	qDLDFevgGa apfNRTHRSK RSSsHPiFhr GEFSVCDSVS VWVgDKTTAT
Orang. NGF	NO:87	qDLDFevgGa apfNRTHRSK RSSsHPiFhr GEFSVCDSVS VWVgDKTTAT
Human NGF	NO:85	qDLDFevgGa apfNRTHRSK RSSsHPiFhr GEFSVCDSVS VWVgDKTTAT
Mouse NGF	NO:89	lDLDFqahGt ipfNRTHRSK RSStHPvFhm GEFSVCDSVS VWVgDKTTAT
Rat NGF	NO:88	lDLDFqahGt isfNRTHRSK RSStHPvFhm GEFSVCDSVS VWVgDKTTAT
Afr. rat NGF	NO:91	lDLDFqahGt isfNRTHRSK RSStHPvFqm GEFSVCDSVS VWVgDKTTAT
G. pig NGF	NO:90	qDLDFevdGa asvNRTHRSK RSStHPvFhm GEFSVCDSVS VWVaDKTTAT
Consensus	NO:84	-DLDF---G- ---NRTHRSK RSS-HP-F-- GEFSVCDSVS VWV-DKTTAT
		151                                                200
Gorilla NGF	NO:86	DIKGkEVmVL gEVNiNNsVF kQYFFETKCR dpnPVdSGCR GIDSKHWNSY
Orang. NGF	NO:87	DIKGkEVmVL gEVNiNNsVF kQYFFETKCR dpnPVdSGCR GIDSKHWNSY
Human NGF	NO:85	DIKGkEVmVL gEVNiNNsVF kQYFFETKCR dpnPVdSGCR GIDSKHWNSY
Mouse NGF	NO:89	DIKGkEVlVL aEVNiNNsVF rQYFFETKCR asnPVeSGCR GIDSKHWNSY
Rat NGF	NO:88	DIKGkEVtVL gEVNiNNsVF kQYFFETKCR apnPVeSGCR GIDSKHWNSY
Aft. rat NGF	NO:91	DIKGnEVlVL gEVNiNNsVF kQYFFETKCR arnPVeSGCR GIDSKHWNSY
G. pig NGF	NO:90	DIKGkEVtVL aEVNvNNnVF kQYFFETKCR dpsPVdSGCR GIDSKHWNSY
Consensus	NO:84	DIKG-EV-VL -EVN-NN-VF -QYFFETKCR ---PV-SGCR GIDSKHWNSY
		201                                                241
Gorilla NGF	NO:86	CTTTHTFVKA LTmdgkQAAW RFIRIDTACV CVLsRKAvRR a
Orang. NGF	NO:87	CTTTHTFVKA LTmdgkQAAW RFIRIDTACV CVLsRKAvRR a
Human NGF	NO:85	CTTTHTFVKA LTmdgkQAAW RFIRIDTACV CVLsRKAvRR a
Mouse NGF	NO:89	CTTTHTFVKA LTtdekQAAW RFIRIDTACV CVLsRKAtRR g
Rat NGF	NO:88	CTTTHTFVKA LTtddkQAAW RFIRIDTACV CVLsRKAaRR g
Aft. rat NGF	NO:91	CTTTHTFVKA LTtddrQAAW RFIRIDTACV CVLtRKApRR g
G. pig NGF	NO:90	CTTTHTFVKA LTtankQAAW RFIRIDTACV CVLnRKAaRR g
Consensus	NO:84	CTTTHTFVKA LT----QAAW RFIRIDTACV CVL-RKA-RR -

TABLE 15
Location of domains within NGF amino acid sequences; numbering
refers to amino acid positions within the corresponding SEQ ID NOs
		Signal		“Mature”A
Polypeptide	SEQ ID	sequence	Pro-peptide	polypeptide
Gorilla NGFB	NO: 86	1-18	19-121	122-241
Orang. NGFB	NO: 87	1-18	19-121	122-241
Human NGF	NO: 85	1-18	19-121	122-241
Mouse NGF	NO: 89	1-18	19-121	122-241
Rat NGF	NO: 88	1-18	19-121	122-241
Afr. rat NGF	NO: 91	1-18	19-121	122-241
G. pig NGF	NO: 90	1-18	19-121	122-241
Consensus	NO: 84	1-18	19-121	122-241
AFor the purposes of the above table, the “mature” polypeptide refers to a polypeptide from which the indicated signal sequence and pro-peptide have been cleaved; additional mature polypeptide forms may occur.
BThe location of the signal sequence, pro-peptide domain, and the mature polypeptide within the gorilla and orangutan NGF amino acid sequences was based on the location of these domains in the amino acid sequences of the other mammalian NGF amino acid sequences.

TABLE 16
Alignment of mammalian BAFF-R amino acid sequences
	SEQ
	ID:	Alignment (numbering based on consensus sequence):
		1                                                   50
Human BAFFR	NO 93	˜˜˜mRrgpRS lRgRDapaPT pCvpaECFDl LVRhCVaCgL lrTprPkpag
Mouse BAFFR	NO:94	mgarRlrvRS qRsRDssvPT qCnqtECFDp LVRnCVsCeL fhT..Pdtgh
Consensus	NO:92	----R---RS -R-RD---PT -C---ECFD- LVR-CV-C-L --T--P---
		51                                                 100
Human BAFFR	NO:93	aSSpaPrTAL QPQEsvgaga GeAalPlpgL LfGAPALLGL aLvLaLV.LV
Mouse BAFFR	NO:94	tSSlePgTAL QPQE...... GsAlrPdvaL LvGAPALLGL iLaLtLVgLV
Consensus	NO:92	-SS--P-TAL QPQE------ G-A--P---L L-GAPALLGL -L-L-LV-LV
		101                                                150
Human BAFFR	NO:93	gLVSWRrRQr rLRgASsaea PDgdkda.pE pLdkViilSp gisdAtAPaW
Mouse BAFFR	NO:94	sLVSWRwRQ. qLRtAS.... PDtsegvqqE sLenVfvpSs etphAsAPtW
Consensus	NO:92	-LVSWR-RQ- -LR-AS---- PD-------E -L--V---S- ----A-AP-W
		151                                    189
Human BAFFR	NO:93	PPpgEDpgtt pPgHSVPVPA TELGSTELVT TKTAGPEQq
Mouse BAFFR	NO:94	PPlkEDadsa lPrHSVPVPA TELGSTELVT TKTAGPEQ˜
Consensus	NO:92	PP--ED---- -P-HSVPVPA TELGSTELVT TKTAGPEQ-

TABLE 17
Location of domains within BAFF-R amino acid sequences; numbering refers
to amino acid positions within the corresponding SEQ ID NOs
	SEQ
Polypeptide	ID	Extracellular	Disulfide bonds	Transmembrane	Intracellular
Human BAFFR	NO: 93	1-76	(19, 32); (24, 35)	77-96	97-184
Mouse BAFFR	NO: 94	1-71	(22, 35); (27, 38)	72-92A	93-175
Consensus	NO: 92	1-79	(22, 35); (27, 38)	80-100	101-189
AThe location of the transmembrane domain in the human BAFF-R amino acid sequence is shown as corresponding to the location of the transmembrane domain in the mouse BAFF-R sequence.

TABLE 18
Alignment of mammalian BAFF amino acid sequences
	SEQ
	ID:	Alignment (numbering based on consensus sequence):
		1                                                   50
Human BAFF	NO:96	MDdSter.eq srLtsClkKr EeMKlkecvs IlPrKEsps. vrsskDGkLL
Mouse BAFF	NO:97	MDeSaktlpp pcLcfCseKg EdMKv.gydp ItPqKEegaw fgicrDGrLL
Consensus	NO:95	MD-S------ --L--C--K- E-MK------ I-P-KE---- -----DG-LL
		51                                                 100
Human BAFF	NO:96	AATLLLALLS cclTvvSfYQ vAALQgDLas LRaELQghha eklPAgAGAP
Mouse BAFF	NO:97	AATLLLALLS ssfTamSlYQ lAALQaDLmn LRmELQsyrg satPAaAGAP
Consensus	NO:95	AATLLLALLS ---T--S-YQ -AALQ-DL-- LR-ELQ---- ---PA-AGAP
		101                                                150
Human BAFF	NO:96	Kagleeapav TAGlKifePp APgegNSSqn sRNkRAvQGP EET.......
Mouse BAFF	NO:97	e........l TAGvKlltPa APrphNSSrg hRNrRAfQGP EETeqdvdls
Consensus	NO:95	---------- TAG-K---P- AP---NSS-- -RN-RA-QGP EET-------
		151                                                200
Human BAFF	NO:96	.......... .......... ....vtQDCL QLIADSeTPT IqKGsYTFVP
Mouse BAFF	NO:97	Appapclpgc rhsqhddngm nlrniiQDCL QLIADSdTPT IrKGtYPFVP
Consensus	NO:95	---------- ---------- ------QDCL QLIADS-TPT I-KG-YTFVP
		201                                                250
Human BAFF	NO:96	WLLSFKRGsA LEEKENKIlV keTGYFFIYg QVLYTDktyA MGHlIQRKKV
Mouse BAFF	NO:97	WLLSFKRGnA LEEKENKIvV rqTGYFFIYs QVLYTDpifA MGHvIQRKKV
Consensus	NO:95	WLLSFKRG-A LEEKENKI-V --TGYFFIY- QVLYTD---A MGH-IQRKRV
		251                                                300
Human BAFF	NO:96	HVFGDELSLV TLFRCIQNMP eTLPNNSCYS AGIAkLEEGD ElQLAIPREN
Mouse BAFF	NO:97	HVFGDELSLV TLFRCIQNMP kTLPNNSCYS AGIArLEEGD EiQLAIPREN
Consensus	NO:95	HVFGDELSLV TLFRCIQNMP -TLPNNSCYS AGIA-LEEGD E-QLAIPREN
		301                318
Human BAFF	NO:96	AQISldGDvT FFGALKLL
Mouse BAFF	NO:97	AQISrnGDdT FFGALKLL
Consensus	NO:95	AQIS--GD-T FFGALKLL

TABLE 19
Location of domains within BAFF amino acid sequences; numbering refers to
amino acid positions within the corresponding SEQ ID NOs
Polypeptide	SEQ ID	Intracellular	Transmembrane	Extracellular	Mature (soluble)A
Human BAFF	NO: 96	1-46	47-67	68-285	134-285
Mouse BAFF	NO: 97	1-47	48-68	69-309	127-309
Consensus	NO: 95	1-48	49-69	70-318	136-318
AFor the purposes of the above table, the “mature” polypeptide refers to an extracellular domain of the polypeptide which has been cleaved from the cell surface to form a soluble polypeptide; other mature forms may occur.

TABLE 20
Alignment of mammalian TNFR1 amino acid sequences
	SEQ
	ID:	Alignment (numbering based on consensus sequence):
Mouse	NO:103	MGLptVPgLL lsLVLlaLLm gihPsgVtGL VpslgDREKR dslCPQGKYv
TNFR1
Rat	NO:102	MGLpiVPgLL lsLVLlaLLm gihPsgVtGL VpslgDREKR dnlCPQGKYa
TNFR1
Cat	NO:100	MGLptVPgLL qpLVLlaLLv eiyPlrVtGL VphlrDREKR aipCPQGKYi
TNFR1
Human	NO:99	MGLstVPdLL lpLVLleLLv giyPsgViGL VphlgDREKR dsvCPQGKYi
TNFR1
Pig	NO:101	MGLstVPgLL lpLVLraLLv dvyPagVhGL VlhpgDREKR eslCPQGKYs
TNFR1
Consensus	NO:98	MGL--VP-LL --LVL--LL- ---P--V-GL V----DREKR ---CPQGKY-
		51                                                   100
Mouse	NO:103	HsknnSICCT KCHKGTYLvs DCpsPGrdTv CreCekGtFT ASqNylrqCL
TNFR1
Rat	NO:102	HpknnSICCT KCHKGTYLvs DCpsPGqeTv CevCdkGtFT ASqNhvrqCL
TNFR1
Cat	NO:100	HpqdnSICCT KCHKGTYLyn DCagPGldTd CreCenGtFT ASeNylrqCL
TNFR1
Human	NO:99	HpqnnSICCT KCHKGTYLyn DCpgPGqdTd CreCesGsFT ASeNhlrhCL
TNFR1
Pig	NO:101	HpqnrSICCT KCHKGTYLhn DClgPGldTd CreCdnGtFT ASeNhltqCL
TNFR1
Consensus	NO:98	H----SICCT KCHKGTYL-- DC--PG--T- C--C--G-FT AS-N----CL
		101                                                 150
Mouse	NO:103	SCktCRkEMs QVEISpCqad kDTVCGCkeN QfqrYlSEth FQCvdCSpCf
TNFR1
Rat	NO:102	SCktCRkEMf QVEISpCkad mDTVCGCkkN QfqrYlSEth FQCvdCSpCf
TNFR1
Cat	NO:100	SCskCRkEMy QVEISpCtvy rDTVCGCrkN QyryYwSEth FQClnCSlCl
TNFR1
Human	NO:99	SCskCRkEMg QVEISsCtvd rDTVCGCrkN QyrhYwSEnl FQCfnCSlCl
TNFR1
Pig	NO:101	SCskCRsEMs QVEISpCtvd rDTVCGCrkN QyrkYwSEtl FQClnCSlCp
TNFR1
Consensus	NO:98	SC--CR-EM- QVEIS-C--- -DTVCGC--N Q---Y-SE-- FQC--CS-C-
		151                                                200
Mouse	NO:103	NGTVtipCkE tQnTvCnCHa GFFLresECv pCshCkKnee CmnkLClpppl
TNFR1
Rat	NO:102	NGTVtipCkE kQnTvCnCHa GFFLsgnECt pCshCkKnqe CmkLCl.ppv
TNFR1
Cat	NO:100	NGTVqisCkE tQnTvCtCHa GFFLrgnECv sCvnCkKnte CtkLCv.piv
TNFR1
Human	NO:99	NGTVhlsCqE kQnTvCtCHa GFFLrenECv sCsnCkKsle CtkLCl.pqi
TNFR1
Pig	NO:101	NGTVqlpClE kQdTiCnCHs GFFLrdkECv sCvnC.Knad CknLCp.ats
TNFR1
Consensus	NO:98	NGTV---C-E -Q-T-C-CH- GFFL---EC- -C--C-K--- C--LC-----
		201                                                250
Mouse	NO:103	anvtnpqDsC TaVLLPLVIl lGlCllsfif isLmcRYprw rpevySIiCr
TNFR1
Rat	NO:102	anvtnpqDsC TaVLLPLVIf lGlCllffic isLlcRYpqw rprvySIiCr
TNFR1
Cat	NO:100	etvkdpqDpG TtVLLPLVIf fGiCvis.fs igLmcRYqrr ksklfSIvCg
TNFR1
Human	NO:99	envkgteDsG TtVLLPLVIf fGlCllsllf igLmyRYqrw ksklySIvCg
TNFR1
Pig	NO:101	etrndfqDtG TtVLLPLVIf fGlClafflf vgLacRYqrw kpklySIiCg
TNFR1
Consensus	NO:98	-------D-G T-VLLPLVI- -G-C------ --L--RY--- -----SI-C-
		251                                                300
Mouse	NO:103	dpvPvKE.Ek ag...kPltp apspaFSPts gfnPTlgFS. tPgfsspvSs
TNFR1
Rat	NO:102	dsapvKEvEg egivtkPltp asipaFSPnp gfnPTlgFSt tPrfshpvSs
TNFR1
Cat	NO:100	kstPtKEgE. ....pqp.l. atgpgFSPip ..sPT..FSp sP..tftpSp
TNFR1
Human	NO:99	kstPeKEgEl egtttkP.l. apnpsFSPtp gftPTlgFSp vPsstftsSs
TNFR1
Pig	NO:101	kstPvKEgEp eplataPsf. gpittFSPip sfsPTttFSp vPsfspisSp
TNFR1
Consensus	NO:98	---P-KE-E- ------P--- -----FSP-- ---PT--FS- -P------S-
		301                                                350
Mouse	NO:103	TpispifgPs nwh.f..mpp vsEvvPt.QG AdPlLyeslc svPaptsvqK
TNFR1
Rat	NO:102	TpispvfgPs nwhnf..vpp vrEvvPt.QG AdPlLygsln pvPipapvrK
TNFR1
Cat	NO:100	T.....ftPs dwanlraasv srEmaPpyQG AgPiLsaapa ssPistpvqK
TNFR1
Human	NO:99	T.....ytPg dcpnf..aap rrEvaPpyQG AdPiLatala sdPipnplqK
TNFR1
Pig	NO:101	T.....ftPc dwsnikvtsp pkEiaPppQG AgPiLpmppa stPvptplpK
TNFR1
Consensus	NO:98	T-------P- ---------- --E--P--QG A-P-L----- --P------K
		351                                                400
Mouse	NO:103	Wed.....sa hPqrpdnaDl AiLYAVVdgV PPaRWKEFmR fmGLSeHEIe
TNFR1
Rat	NO:102	Wed...vvaa qPgrldtaDp AmLYAVVdgV PPtRWKEFmR llGLSeHEIe
TNFR1
Cat	NO:100	Wedstht..q rPea.dpaDp AtLYAVVdgV PPsRWKEFvR rlGLSeHEIe
TNFR1
Human	NO:99	Wedsah.... kPqsldtdDp AtLYAVVenV PPlRWKEFvR rlGLSdHEId
TNFR1
Pig	NO:101	Wggsahsahs aPaqladaDp AtLYAVVdgV PPtRWKEFvR rlGLSeHEIe
TNFR1
Consensus	NO:98	W--------- -P------D- A-LYAVV--V PP-RWKEF-R --GLS-HEI-
		401                                                450
Mouse	NO:103	RLEmQNGRCL REAqYSMLea WRRRTpRhEd TLevVGlVLs kMnLaGCLEn
TNFR1
Rat	NO:102	RLElQNGRCL REAhYSMLea WRRRTpRhEa TLdvvGrVLc dMnLrGCLEn
TNFR1
Cat	NO:100	RLElQNGRCL REAhYSMLaa WRRRTpRrEa TLellGrVLr dMdLlGCLEd
TNFR1
Human	NO:99	RLElQNGRCL REAqYSMLat WRRRTpRrEa TLellGrVLr dMdLlGCLEd
TNFR1
Pig	NO:101	RLElQNGRCL REAcYSMLae WRRRTsRrEa TLellGsVLr dMdLlGCLEd
TNFR1
Consensus	NO:98	RLE-QNGRCL REA-YSML-- WRRRT-R-E- TL---G-VL- -M-L-GCLE-
		451                 469
Mouse	NO:103	IlEaLrnPAp .ssttrLpR
TNFR1
Rat	NO:102	IrEtLesPAh .sstthLpR
TNFR1
Cat	NO:100	IeEaLcaPAs lspaprLlR
TNFR1
Human	NO:99	IeEaLcgPAa lppapsLlR
TNFR1
Pig	NO:101	IeEaLrgPAr lapaphLlR
TNFR1
Consensus	NO:98	I-E-L--PA- ------L-R

TABLE 21
Location of domains within TNFR1 amino acid sequences; numbering refers
to amino acid positions within the corresponding SEQ ID NOs
	SEQ					“Mature”A
Polypeptide	ID	Signal	ExtracellularS	Transmembrane	Intracellular	polypeptide
Mouse TNFR1	NO: 103	1-21	22-212	213-235	236-454	22-454
Rat TNFR1	NO: 102	1-21	22-211	212-234	235-461	22-461
Cat TNFR1B	NO: 100	1-21	22-211	212-233	234-446	22-446
Human TNFR1	NO: 99	1-21	22-211	212-234	235-455	22-455
Pig TNFR1	NO: 101	1-21	22-210	211-233	234-461	22-461
Consensus	NO: 98	1-21	22-212	213-235	236-469	22-469
AFor the purposes of the above table, the “mature” polypeptide refers to a polypeptide from which the indicated signal sequence has been cleaved; additional mature polypeptide forms may occur.
BThe positions of domains within the cat TNFR1 amino acid sequence have been determined by comparison with the corresponding domains of the other mammalian TNFR1 amino acid sequences.

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

1. A method for detecting the presence of a compound in a sample, comprising:

(a) providing, in any order: (i) a sample suspected of comprising a compound and a control sample without the compound; (ii) a receptor and a response gene; and (iii) a ligand, wherein the ligand is capable of binding the receptor, thereby altering the expression of the response gene;

(b) combining, in any order, (i) the sample, the receptor, and the ligand;

and (ii) the control sample, the receptor and the ligand; and

(c) measuring the level of the expression of the response gene;

wherein the presence of the compound in the sample is detected by an alteration in the level of expression of the response gene when compared to the level of expression of the response gene when the receptor is combined with the ligand in the presence of the control sample.

2. A method for detecting the presence of a compound in the presence or absence of a sample, comprising:

(a) providing, in any order: (i) a compound, wherein the compound is in the presence or absence of a sample; (ii) a receptor and a response gene; and (iii) a ligand, wherein the ligand is capable of binding the receptor, thereby altering the expression of the response gene;

(b) combining, in any order, (i) the compound, the receptor, and the ligand; and (ii) the receptor and the ligand; and

(c) measuring the level of the expression of the response gene, wherein the presence of the compound is measured by an alteration in the level of expression of the response gene when the receptor is combined with the ligand and the compound compared to the level of expression of the response gene when the receptor is combined with the ligand only; and wherein when the receptor is combined with varying concentrations of the ligand and the compound, the expression of the response gene in the presence of the sample is correlated with the expression of the response gene in the absence of the sample with a correlation coefficient of at least 0.5.

3. The method of claim 1, wherein the ligand is a therapeutic substance for administration to a subject.

4. The method of claim 3, wherein the compound is a neutralizing antibody against the therapeutic substance.

5. The method of any one of claims 1-4, wherein the receptor comprises SEQ ID NO:1.

6. The method of any one of claims 1-4, wherein the receptor comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.

7. The method of claim 5, wherein the therapeutic substance comprises SEQ ID NO:6.

8. The method of claim 6, wherein the therapeutic substance comprises SEQ ID NO:6.

9. The method of claim 5, wherein the therapeutic substance comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

10. The method of claim 6, wherein the therapeutic substance comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

11. The method of claim 5, wherein the response gene comprises SEQ ID NO:15.

12. The method of claim 6, wherein the response gene comprises SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

13. The method of any one of claims 1-4, wherein the receptor comprises the extracellular domain of SEQ ID NO:80.

14. The method of any one of claims 1-4, wherein the receptor comprises the extracellular domain of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

15. The method of claim 13, wherein the ligand comprises SEQ ID NO:84.

16. The method of claim 14, wherein the ligand comprises SEQ ID NO:84.

17. The method of claim 13, wherein the ligand comprises SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:91.

18. The method of claim 14, wherein the ligand comprises SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, or SEQ ID NO:91.

19. The method of claim 13, wherein the response gene comprises SEQ ID NO:15.

20. The method of claim 14, wherein the response gene comprises SEQ ID NO:15.

21. The method of any one of claims 1-4, wherein the receptor comprises the extracellular domain of SEQ ID NO:92.

22. The method of any one of claims 1-4, wherein the receptor comprises the extracellular domain of SEQ ID NO:93 or SEQ ID NO:94.

23. The method of claim 21, wherein the ligand comprises SEQ ID NO:95.

24. The method of claim 22, wherein the ligand comprises SEQ ID NO:95.

25. The method of claim 21, wherein the ligand comprises SEQ ID NO:96 or SEQ ID NO:97.

26. The method of claim 22, wherein the ligand comprises SEQ ID NO:96 or SEQ ID NO:97.

27. The method of claim 21, wherein the response gene comprises SEQ ID NO:98.

28. The method of claim 22, wherein the response gene comprises SEQ ID NO:98.

28. The method of claim 21, wherein the response gene comprises SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:103.

29. The method of claim 23, wherein the response gene comprises SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, or SEQ ID NO:103.

30. The method of claim 21, wherein the response gene comprises SEQ ID NO:15.

31. The method of claim 22, wherein the response gene comprises SEQ ID NO:15.

32. The method of claim 23, wherein the response gene comprises SEQ ID NO:15.

33. The method of claim 24, wherein the response gene comprises SEQ ID NO:15.

34. The method of any one of claims 1-4, wherein the ligand comprises SEQ ID NO:105, SEQ ID NO:106, or SEQ ID NO:107.

35. The method of claim 34, wherein the receptor comprises SEQ ID NO:108 or SEQ ID NO:109.

36. The method of claim 34, wherein the response gene is tartrate resistant acid phosphatase (TRAP).

37. The method of any one of claims 1-4, wherein the ligand is an endogenous ligand, which is bound by a therapeutic substance for administration to a subject.

38. The method of any one of claims 1-4, wherein the level of the expression of the response gene is measured using a bDNA assay.

39. The method of any of the claims 1-4, wherein the sample is selected from the group consisting of whole blood, plasma, serum, synovial fluid, ascitic fluid, lacrimal fluid, perspiration, seminal fluid, cell extracts, and tissue extracts.

40. The method any one of claims 1-4, wherein the receptor is expressed by a mammalian cell.

41. A kit comprising (a) a cell expressing a receptor, wherein the receptor comprises the intracellular domain of EPOR, and (b) one or more oligonucleotides used to detect PIM 1 gene expression, the oligonucleotides selected from the group consisting of SEQ ID NOs:22 through 79.